US20120004457A1 - Method for producing peptide - Google Patents
Method for producing peptide Download PDFInfo
- Publication number
- US20120004457A1 US20120004457A1 US13/122,850 US200913122850A US2012004457A1 US 20120004457 A1 US20120004457 A1 US 20120004457A1 US 200913122850 A US200913122850 A US 200913122850A US 2012004457 A1 US2012004457 A1 US 2012004457A1
- Authority
- US
- United States
- Prior art keywords
- group
- peptide
- producing
- fmoc
- thiol
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 162
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 30
- 150000007970 thio esters Chemical group 0.000 claims abstract description 34
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 claims abstract description 29
- 150000003587 threonine derivatives Chemical class 0.000 claims abstract description 28
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 claims abstract description 27
- 210000004899 c-terminal region Anatomy 0.000 claims abstract description 24
- 150000003862 amino acid derivatives Chemical class 0.000 claims abstract description 18
- 239000003638 chemical reducing agent Substances 0.000 claims abstract description 17
- 150000003573 thiols Chemical class 0.000 claims description 58
- -1 phosphine compound Chemical class 0.000 claims description 25
- 125000004432 carbon atom Chemical group C* 0.000 claims description 20
- 125000004435 hydrogen atom Chemical group [H]* 0.000 claims description 20
- 125000001570 methylene group Chemical group [H]C([H])([*:1])[*:2] 0.000 claims description 20
- 150000001875 compounds Chemical class 0.000 claims description 19
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 claims description 18
- 125000006239 protecting group Chemical group 0.000 claims description 17
- XYFCBTPGUUZFHI-UHFFFAOYSA-N Phosphine Natural products P XYFCBTPGUUZFHI-UHFFFAOYSA-N 0.000 claims description 15
- 125000000217 alkyl group Chemical group 0.000 claims description 13
- 125000003277 amino group Chemical group 0.000 claims description 11
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 10
- 125000000999 tert-butyl group Chemical group [H]C([H])([H])C(*)(C([H])([H])[H])C([H])([H])[H] 0.000 claims description 10
- 229910000073 phosphorus hydride Inorganic materials 0.000 claims description 9
- 125000003118 aryl group Chemical group 0.000 claims description 6
- 238000000034 method Methods 0.000 abstract description 52
- 238000006243 chemical reaction Methods 0.000 abstract description 45
- 239000000126 substance Substances 0.000 abstract description 12
- 125000003396 thiol group Chemical class [H]S* 0.000 abstract description 12
- 238000007086 side reaction Methods 0.000 abstract description 7
- 125000000524 functional group Chemical group 0.000 abstract description 5
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 39
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 37
- 239000000047 product Substances 0.000 description 33
- 239000011347 resin Substances 0.000 description 29
- 229920005989 resin Polymers 0.000 description 29
- 239000000243 solution Substances 0.000 description 29
- 125000003088 (fluoren-9-ylmethoxy)carbonyl group Chemical group 0.000 description 24
- 239000000203 mixture Substances 0.000 description 24
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 23
- 238000003786 synthesis reaction Methods 0.000 description 22
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 21
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 description 21
- 230000015572 biosynthetic process Effects 0.000 description 21
- SECXISVLQFMRJM-UHFFFAOYSA-N N-Methylpyrrolidone Chemical compound CN1CCCC1=O SECXISVLQFMRJM-UHFFFAOYSA-N 0.000 description 17
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 16
- DTQVDTLACAAQTR-UHFFFAOYSA-N trifluoroacetic acid Substances OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 16
- QOSSAOTZNIDXMA-UHFFFAOYSA-N Dicylcohexylcarbodiimide Chemical compound C1CCCCC1N=C=NC1CCCCC1 QOSSAOTZNIDXMA-UHFFFAOYSA-N 0.000 description 14
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 13
- 239000002244 precipitate Substances 0.000 description 13
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 12
- PZBFGYYEXUXCOF-UHFFFAOYSA-N TCEP Chemical compound OC(=O)CCP(CCC(O)=O)CCC(O)=O PZBFGYYEXUXCOF-UHFFFAOYSA-N 0.000 description 12
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 11
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine (DIPEA) Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 description 11
- 239000008055 phosphate buffer solution Substances 0.000 description 11
- MUSGYEMSJUFFHT-UWABRSFTSA-N 2-[(4R,7S,10S,13S,19S,22S,25S,28S,31S,34R)-34-[[(2S,3S)-2-[[(2R)-2-amino-3-(4-hydroxyphenyl)propanoyl]amino]-3-methylpentanoyl]amino]-4-[[(2S,3S)-1-amino-3-methyl-1-oxopentan-2-yl]-methylcarbamoyl]-25-(3-amino-3-oxopropyl)-7-(3-carbamimidamidopropyl)-10-(1H-imidazol-5-ylmethyl)-19-(1H-indol-3-ylmethyl)-13,17-dimethyl-28-[(1-methylindol-3-yl)methyl]-6,9,12,15,18,21,24,27,30,33-decaoxo-31-propan-2-yl-1,2-dithia-5,8,11,14,17,20,23,26,29,32-decazacyclopentatriacont-22-yl]acetic acid Chemical compound CC[C@H](C)[C@H](NC(=O)[C@H](N)Cc1ccc(O)cc1)C(=O)N[C@H]1CSSC[C@H](NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](Cc2cnc[nH]2)NC(=O)[C@H](C)NC(=O)CN(C)C(=O)[C@H](Cc2c[nH]c3ccccc23)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](Cc2cn(C)c3ccccc23)NC(=O)[C@@H](NC1=O)C(C)C)C(=O)N(C)[C@@H]([C@@H](C)CC)C(N)=O MUSGYEMSJUFFHT-UWABRSFTSA-N 0.000 description 10
- HEDRZPFGACZZDS-MICDWDOJSA-N Trichloro(2H)methane Chemical compound [2H]C(Cl)(Cl)Cl HEDRZPFGACZZDS-MICDWDOJSA-N 0.000 description 10
- NPZTUJOABDZTLV-UHFFFAOYSA-N hydroxybenzotriazole Substances O=C1C=CC=C2NNN=C12 NPZTUJOABDZTLV-UHFFFAOYSA-N 0.000 description 10
- 238000010898 silica gel chromatography Methods 0.000 description 10
- QYIGFZOHYGYBLX-UHFFFAOYSA-N 2-phenyl-2-sulfanylacetic acid Chemical compound OC(=O)C(S)C1=CC=CC=C1 QYIGFZOHYGYBLX-UHFFFAOYSA-N 0.000 description 9
- 238000000746 purification Methods 0.000 description 9
- WFDIJRYMOXRFFG-UHFFFAOYSA-N Acetic anhydride Chemical compound CC(=O)OC(C)=O WFDIJRYMOXRFFG-UHFFFAOYSA-N 0.000 description 8
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 8
- 239000007853 buffer solution Substances 0.000 description 8
- HNKJADCVZUBCPG-UHFFFAOYSA-N thioanisole Chemical compound CSC1=CC=CC=C1 HNKJADCVZUBCPG-UHFFFAOYSA-N 0.000 description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 8
- BWGNESOTFCXPMA-UHFFFAOYSA-N Dihydrogen disulfide Chemical compound SS BWGNESOTFCXPMA-UHFFFAOYSA-N 0.000 description 7
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical class [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 7
- 239000011259 mixed solution Substances 0.000 description 7
- 239000002904 solvent Substances 0.000 description 7
- 238000005160 1H NMR spectroscopy Methods 0.000 description 6
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 6
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- NQRYJNQNLNOLGT-UHFFFAOYSA-N Piperidine Chemical compound C1CCNCC1 NQRYJNQNLNOLGT-UHFFFAOYSA-N 0.000 description 6
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- 239000003795 chemical substances by application Substances 0.000 description 6
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 6
- 239000003960 organic solvent Substances 0.000 description 6
- 238000000926 separation method Methods 0.000 description 6
- 150000003354 serine derivatives Chemical class 0.000 description 6
- 239000007858 starting material Substances 0.000 description 6
- RMVRSNDYEFQCLF-UHFFFAOYSA-N thiophenol Chemical compound SC1=CC=CC=C1 RMVRSNDYEFQCLF-UHFFFAOYSA-N 0.000 description 6
- DQJCDTNMLBYVAY-ZXXIYAEKSA-N (2S,5R,10R,13R)-16-{[(2R,3S,4R,5R)-3-{[(2S,3R,4R,5S,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy}-5-(ethylamino)-6-hydroxy-2-(hydroxymethyl)oxan-4-yl]oxy}-5-(4-aminobutyl)-10-carbamoyl-2,13-dimethyl-4,7,12,15-tetraoxo-3,6,11,14-tetraazaheptadecan-1-oic acid Chemical compound NCCCC[C@H](C(=O)N[C@@H](C)C(O)=O)NC(=O)CC[C@H](C(N)=O)NC(=O)[C@@H](C)NC(=O)C(C)O[C@@H]1[C@@H](NCC)C(O)O[C@H](CO)[C@H]1O[C@H]1[C@H](NC(C)=O)[C@@H](O)[C@H](O)[C@@H](CO)O1 DQJCDTNMLBYVAY-ZXXIYAEKSA-N 0.000 description 5
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 5
- 0 C.C.[1*]C(O[2*]SC)C(NC)C(=O)O Chemical compound C.C.[1*]C(O[2*]SC)C(NC)C(=O)O 0.000 description 5
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical class [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 5
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 5
- 235000018417 cysteine Nutrition 0.000 description 5
- 238000010511 deprotection reaction Methods 0.000 description 5
- 239000012153 distilled water Substances 0.000 description 5
- 238000001840 matrix-assisted laser desorption--ionisation time-of-flight mass spectrometry Methods 0.000 description 5
- 102000002068 Glycopeptides Human genes 0.000 description 4
- 108010015899 Glycopeptides Proteins 0.000 description 4
- 102000003886 Glycoproteins Human genes 0.000 description 4
- 108090000288 Glycoproteins Proteins 0.000 description 4
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 4
- 125000000539 amino acid group Chemical group 0.000 description 4
- 125000003710 aryl alkyl group Chemical group 0.000 description 4
- 125000004429 atom Chemical group 0.000 description 4
- 125000001797 benzyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])* 0.000 description 4
- 239000006227 byproduct Substances 0.000 description 4
- 239000003153 chemical reaction reagent Substances 0.000 description 4
- 238000006477 desulfuration reaction Methods 0.000 description 4
- 230000023556 desulfurization Effects 0.000 description 4
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 4
- 238000004128 high performance liquid chromatography Methods 0.000 description 4
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- RUDNWZFWWJFUSF-UHFFFAOYSA-M potassium;(4-methylphenyl)-oxido-oxo-sulfanylidene-$l^{6}-sulfane Chemical compound [K+].CC1=CC=C(S([O-])(=O)=S)C=C1 RUDNWZFWWJFUSF-UHFFFAOYSA-M 0.000 description 4
- 102000004196 processed proteins & peptides Human genes 0.000 description 4
- 238000004007 reversed phase HPLC Methods 0.000 description 4
- 229910052938 sodium sulfate Inorganic materials 0.000 description 4
- 235000011152 sodium sulphate Nutrition 0.000 description 4
- GLGNXYJARSMNGJ-VKTIVEEGSA-N (1s,2s,3r,4r)-3-[[5-chloro-2-[(1-ethyl-6-methoxy-2-oxo-4,5-dihydro-3h-1-benzazepin-7-yl)amino]pyrimidin-4-yl]amino]bicyclo[2.2.1]hept-5-ene-2-carboxamide Chemical compound CCN1C(=O)CCCC2=C(OC)C(NC=3N=C(C(=CN=3)Cl)N[C@H]3[C@H]([C@@]4([H])C[C@@]3(C=C4)[H])C(N)=O)=CC=C21 GLGNXYJARSMNGJ-VKTIVEEGSA-N 0.000 description 3
- WMSUFWLPZLCIHP-UHFFFAOYSA-N (2,5-dioxopyrrolidin-1-yl) 9h-fluoren-9-ylmethyl carbonate Chemical compound C12=CC=CC=C2C2=CC=CC=C2C1COC(=O)ON1C(=O)CCC1=O WMSUFWLPZLCIHP-UHFFFAOYSA-N 0.000 description 3
- UTQHLYJFMFKSGI-UBINZTMLSA-N (2s)-2-[[(2s,3s)-2-[[(2s)-2-[[(2s)-1-[(2s)-2-[[(2s)-2-[[(2s,3r)-3-[(2s,4r,5r,6r)-3-acetamido-5-hydroxy-6-(hydroxymethyl)-4-[(2r,3r,4s,5r,6r)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxyoxan-2-yl]oxy-2-[2-[[(2s)-4-amino-2-[[(2s)-2-[[2-[[2-[[(2s)-4-carbo Chemical compound C([C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(C)C)C(O)=O)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CCCCN)NC(=O)[C@H](CCCCN)NC(=O)[C@@H](NC(=O)C(C)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CO)NC(=O)CNC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@H]1NC(=O)CC1)[C@@H](C)O[C@@H]1C([C@@H](O[C@H]2[C@@H]([C@@H](O)[C@@H](O)[C@@H](CO)O2)O)[C@@H](O)[C@@H](CO)O1)NC(C)=O)C1=CC=C(O)C=C1 UTQHLYJFMFKSGI-UBINZTMLSA-N 0.000 description 3
- ONBQEOIKXPHGMB-VBSBHUPXSA-N 1-[2-[(2s,3r,4s,5r)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]oxy-4,6-dihydroxyphenyl]-3-(4-hydroxyphenyl)propan-1-one Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1OC1=CC(O)=CC(O)=C1C(=O)CCC1=CC=C(O)C=C1 ONBQEOIKXPHGMB-VBSBHUPXSA-N 0.000 description 3
- JMTMSDXUXJISAY-UHFFFAOYSA-N 2H-benzotriazol-4-ol Chemical compound OC1=CC=CC2=C1N=NN2 JMTMSDXUXJISAY-UHFFFAOYSA-N 0.000 description 3
- 101710156565 Contulakin-G Proteins 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- 229910020889 NaBH3 Inorganic materials 0.000 description 3
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 description 3
- 239000002253 acid Substances 0.000 description 3
- 230000002378 acidificating effect Effects 0.000 description 3
- 150000001408 amides Chemical class 0.000 description 3
- 235000001014 amino acid Nutrition 0.000 description 3
- 150000001413 amino acids Chemical class 0.000 description 3
- 238000007098 aminolysis reaction Methods 0.000 description 3
- 125000000484 butyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 3
- 239000007795 chemical reaction product Substances 0.000 description 3
- 229940125758 compound 15 Drugs 0.000 description 3
- 229940126142 compound 16 Drugs 0.000 description 3
- 238000009833 condensation Methods 0.000 description 3
- 230000005494 condensation Effects 0.000 description 3
- 238000007796 conventional method Methods 0.000 description 3
- 230000007547 defect Effects 0.000 description 3
- 229910001873 dinitrogen Inorganic materials 0.000 description 3
- 238000001035 drying Methods 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 125000004051 hexyl group Chemical group [H]C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])* 0.000 description 3
- 238000002000 high resolution fast-atom bombardment mass spectrometry Methods 0.000 description 3
- 125000001147 pentyl group Chemical group C(CCCC)* 0.000 description 3
- COLNVLDHVKWLRT-QMMMGPOBSA-N phenylalanine group Chemical group N[C@@H](CC1=CC=CC=C1)C(=O)O COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 description 3
- 125000001436 propyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])[H] 0.000 description 3
- 235000018102 proteins Nutrition 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 239000012279 sodium borohydride Substances 0.000 description 3
- 229910000033 sodium borohydride Inorganic materials 0.000 description 3
- YBBRCQOCSYXUOC-UHFFFAOYSA-N sulfuryl dichloride Chemical compound ClS(Cl)(=O)=O YBBRCQOCSYXUOC-UHFFFAOYSA-N 0.000 description 3
- 230000002194 synthesizing effect Effects 0.000 description 3
- WMXCDAVJEZZYLT-UHFFFAOYSA-N tert-butylthiol Chemical compound CC(C)(C)S WMXCDAVJEZZYLT-UHFFFAOYSA-N 0.000 description 3
- 238000011282 treatment Methods 0.000 description 3
- GHYOCDFICYLMRF-UTIIJYGPSA-N (2S,3R)-N-[(2S)-3-(cyclopenten-1-yl)-1-[(2R)-2-methyloxiran-2-yl]-1-oxopropan-2-yl]-3-hydroxy-3-(4-methoxyphenyl)-2-[[(2S)-2-[(2-morpholin-4-ylacetyl)amino]propanoyl]amino]propanamide Chemical compound C1(=CCCC1)C[C@@H](C(=O)[C@@]1(OC1)C)NC([C@H]([C@@H](C1=CC=C(C=C1)OC)O)NC([C@H](C)NC(CN1CCOCC1)=O)=O)=O GHYOCDFICYLMRF-UTIIJYGPSA-N 0.000 description 2
- QFLWZFQWSBQYPS-AWRAUJHKSA-N (3S)-3-[[(2S)-2-[[(2S)-2-[5-[(3aS,6aR)-2-oxo-1,3,3a,4,6,6a-hexahydrothieno[3,4-d]imidazol-4-yl]pentanoylamino]-3-methylbutanoyl]amino]-3-(4-hydroxyphenyl)propanoyl]amino]-4-[1-bis(4-chlorophenoxy)phosphorylbutylamino]-4-oxobutanoic acid Chemical compound CCCC(NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](Cc1ccc(O)cc1)NC(=O)[C@@H](NC(=O)CCCCC1SC[C@@H]2NC(=O)N[C@H]12)C(C)C)P(=O)(Oc1ccc(Cl)cc1)Oc1ccc(Cl)cc1 QFLWZFQWSBQYPS-AWRAUJHKSA-N 0.000 description 2
- IWZSHWBGHQBIML-ZGGLMWTQSA-N (3S,8S,10R,13S,14S,17S)-17-isoquinolin-7-yl-N,N,10,13-tetramethyl-2,3,4,7,8,9,11,12,14,15,16,17-dodecahydro-1H-cyclopenta[a]phenanthren-3-amine Chemical compound CN(C)[C@H]1CC[C@]2(C)C3CC[C@@]4(C)[C@@H](CC[C@@H]4c4ccc5ccncc5c4)[C@@H]3CC=C2C1 IWZSHWBGHQBIML-ZGGLMWTQSA-N 0.000 description 2
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 2
- XTHFKEDIFFGKHM-UHFFFAOYSA-N Dimethoxyethane Chemical compound COCCOC XTHFKEDIFFGKHM-UHFFFAOYSA-N 0.000 description 2
- FFFHZYDWPBMWHY-VKHMYHEASA-N L-homocysteine Chemical compound OC(=O)[C@@H](N)CCS FFFHZYDWPBMWHY-VKHMYHEASA-N 0.000 description 2
- AYFVYJQAPQTCCC-GBXIJSLDSA-N L-threonine Chemical compound C[C@@H](O)[C@H](N)C(O)=O AYFVYJQAPQTCCC-GBXIJSLDSA-N 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 2
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 description 2
- 239000004473 Threonine Substances 0.000 description 2
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 2
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- 238000005406 washing Methods 0.000 description 2
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- 125000006181 4-methyl benzyl group Chemical group [H]C1=C([H])C(=C([H])C([H])=C1C([H])([H])[H])C([H])([H])* 0.000 description 1
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- OVRNDRQMDRJTHS-KEWYIRBNSA-N N-acetyl-D-galactosamine Chemical compound CC(=O)N[C@H]1C(O)O[C@H](CO)[C@H](O)[C@@H]1O OVRNDRQMDRJTHS-KEWYIRBNSA-N 0.000 description 1
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- 229910006024 SO2Cl2 Inorganic materials 0.000 description 1
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 1
- WXIONIWNXBAHRU-UHFFFAOYSA-N [dimethylamino(triazolo[4,5-b]pyridin-3-yloxy)methylidene]-dimethylazanium Chemical compound C1=CN=C2N(OC(N(C)C)=[N+](C)C)N=NC2=C1 WXIONIWNXBAHRU-UHFFFAOYSA-N 0.000 description 1
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- JMXMXKRNIYCNRV-UHFFFAOYSA-N bis(hydroxymethyl)phosphanylmethanol Chemical compound OCP(CO)CO JMXMXKRNIYCNRV-UHFFFAOYSA-N 0.000 description 1
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- HDFRDWFLWVCOGP-UHFFFAOYSA-N carbonothioic O,S-acid Chemical compound OC(S)=O HDFRDWFLWVCOGP-UHFFFAOYSA-N 0.000 description 1
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- CCAFPWNGIUBUSD-UHFFFAOYSA-N diethyl sulfoxide Chemical compound CCS(=O)CC CCAFPWNGIUBUSD-UHFFFAOYSA-N 0.000 description 1
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 description 1
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- 238000001914 filtration Methods 0.000 description 1
- 238000001641 gel filtration chromatography Methods 0.000 description 1
- 229960000789 guanidine hydrochloride Drugs 0.000 description 1
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- 229910052736 halogen Inorganic materials 0.000 description 1
- 230000002140 halogenating effect Effects 0.000 description 1
- 150000002367 halogens Chemical class 0.000 description 1
- 125000003187 heptyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 125000001972 isopentyl group Chemical group [H]C([H])([H])C([H])(C([H])([H])[H])C([H])([H])C([H])([H])* 0.000 description 1
- ZLVXBBHTMQJRSX-VMGNSXQWSA-N jdtic Chemical compound C1([C@]2(C)CCN(C[C@@H]2C)C[C@H](C(C)C)NC(=O)[C@@H]2NCC3=CC(O)=CC=C3C2)=CC=CC(O)=C1 ZLVXBBHTMQJRSX-VMGNSXQWSA-N 0.000 description 1
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- 239000002858 neurotransmitter agent Substances 0.000 description 1
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- 239000000825 pharmaceutical preparation Substances 0.000 description 1
- 229940127557 pharmaceutical product Drugs 0.000 description 1
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- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 230000006340 racemization Effects 0.000 description 1
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- 230000035484 reaction time Effects 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 229910052709 silver Inorganic materials 0.000 description 1
- 239000004332 silver Substances 0.000 description 1
- 229910000029 sodium carbonate Inorganic materials 0.000 description 1
- 229910052717 sulfur Inorganic materials 0.000 description 1
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- 125000003944 tolyl group Chemical group 0.000 description 1
- 230000007704 transition Effects 0.000 description 1
- TUQOTMZNTHZOKS-UHFFFAOYSA-N tributylphosphine Chemical compound CCCCP(CCCC)CCCC TUQOTMZNTHZOKS-UHFFFAOYSA-N 0.000 description 1
- RXJKFRMDXUJTEX-UHFFFAOYSA-N triethylphosphine Chemical compound CCP(CC)CC RXJKFRMDXUJTEX-UHFFFAOYSA-N 0.000 description 1
- IGNTWNVBGLNYDV-UHFFFAOYSA-N triisopropylphosphine Chemical compound CC(C)P(C(C)C)C(C)C IGNTWNVBGLNYDV-UHFFFAOYSA-N 0.000 description 1
- 125000002221 trityl group Chemical group [H]C1=C([H])C([H])=C([H])C([H])=C1C([*])(C1=C(C(=C(C(=C1[H])[H])[H])[H])[H])C1=C([H])C([H])=C([H])C([H])=C1[H] 0.000 description 1
- 125000005023 xylyl group Chemical group 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/02—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length in solution
- C07K1/026—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length in solution by fragment condensation in solution
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
- C07B51/00—Introduction of protecting groups or activating groups, not provided for in the preceding groups
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C323/00—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups
- C07C323/10—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and singly-bound oxygen atoms bound to the same carbon skeleton
- C07C323/11—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and singly-bound oxygen atoms bound to the same carbon skeleton having the sulfur atoms of the thio groups bound to acyclic carbon atoms of the carbon skeleton
- C07C323/12—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and singly-bound oxygen atoms bound to the same carbon skeleton having the sulfur atoms of the thio groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being acyclic and saturated
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C2603/00—Systems containing at least three condensed rings
- C07C2603/02—Ortho- or ortho- and peri-condensed systems
- C07C2603/04—Ortho- or ortho- and peri-condensed systems containing three rings
- C07C2603/06—Ortho- or ortho- and peri-condensed systems containing three rings containing at least one ring with less than six ring members
- C07C2603/10—Ortho- or ortho- and peri-condensed systems containing three rings containing at least one ring with less than six ring members containing five-membered rings
- C07C2603/12—Ortho- or ortho- and peri-condensed systems containing three rings containing at least one ring with less than six ring members containing five-membered rings only one five-membered ring
- C07C2603/18—Fluorenes; Hydrogenated fluorenes
Definitions
- the present invention relates to a novel method for synthesizing a peptide. More specifically, the present invention relates to a novel method for synthesizing a peptide utilizing a serine or threonine derivative having a thiol auxiliary group, and an amino acid derivative having a thioester group.
- Non-Patent Document 1 a thioester method in which peptide thioester segments prepared by a solid-phase method are repeatedly condensed
- Non-Patent Document 2 a native chemical ligation method
- the thioester method realizes a segment-to-segment condensation by activating, with silver ions, the thioester terminal groups of the partially protected peptide thioester.
- an intermolecular thioester bond is formed between a thioester terminal group of a peptide thioester having no protecting group and a cysteine residue at an N-terminal end of another peptide, followed by formation of a peptide bond by an intermolecular aminolysis reaction of the amino group of the cysteine residue ( FIG. 1 ).
- the native chemical ligation method is more convenient than the thioester method because the peptide prepared by the solid-phase method can be used as it is in the condensation. Accordingly, the native chemical ligation method is becoming the mainstream of a chemical synthesis of a protein.
- the native chemical ligation method however, has a defect in which condensation can be performed only between peptide thioester having a thioester terminal group but no protecting group and a peptide having cysteine at the N-terminal end.
- the native chemical ligation methods using the thiol auxiliary group include, for example, a method in which after ligation of cysteine is performed, the resulting cysteine is converted into alanine through a desulfurization reaction (Non-Patent Documents 3-5); a method in which after ligation is performed by using a phenylalanine residue having a thiol auxiliary group at the ⁇ -potision, the conversion into phenylalanine is performed through a desulfurization reaction (Non-Patent Document 6); a method in which after ligation of homocysteine is performed, conversion into a methionine residue is performed through methylation (Non-Patent Document 7); a method in which a thiol auxiliary group is bonded to an acetamido group or hydroxyl
- Non-Patent Documents 8-12 a method in which a thiol auxiliary group is bonded to a side-chain carboxyl group, and the ligation is performed (Non-Patent Document 13), and the like.
- Non-Patent Documents 3-5 and 6 the peptide is adsorbed on a metal, thus resulting in the lowering of a recovery rate, because of the use of a metal catalyst in the desulfurization reaction, and, furthermore, side reactions such as demethylthiolation in the methionine residue of the peptide may occur.
- Non-Patent Documents 8-12 and 13 when the acetamido group is used, the same situations as described above result because the desulfurization reaction is finally performed, and when the sugar chain hydroxyl group is used, ⁇ -elimination of the sugar chain or racemization of ⁇ -carbon may possibly occur because an alkaline treatment is finally performed for the removal.
- the efficiency of the ligation is low, and side reactions to the functional groups in the various amino acid residues in the peptide may possibly occur.
- An object to be solved by the present invention is to solve the problems of the prior art described above. That is, the object to be solved by the present invention is to provide a novel method for producing a peptide utilizing a ligation reaction in which ligation efficiency is excellent and side reactions to other functional groups in the peptide are hard to occur, in comparison with the conventional native chemical ligation methods utilizing the thiol auxiliary group. Furthermore, a second object to be solved by the present invention is to provide a compound having a novel thiol auxiliary group to be used in the production method described above.
- the present inventors have found that when a first peptide containing an amino acid derivative having a thioester group and a second peptide containing a serine or threonine derivative having a thiol auxiliary group are caused to react in the presence of a reducing agent, a ligated product of the first peptide and the second peptide can be obtained with high ligation efficiency and without the occurrence of side reaction to other functional groups in the peptide; and have reached the accomplishment of the present invention.
- the present invention provides a method for producing a peptide which comprises a step of causing a first peptide and a second peptide to react in the presence of a reducing agent to obtain a ligated product of the first peptide and the second peptide, wherein the first peptide contains, at the C-terminal end, an amino acid derivative having a thioester group, and the second peptide contains, at the N-terminal end, a serine or threonine derivative having a thiol auxiliary group.
- the thiol auxiliary group is a group containing a disulfide bond.
- the reducing agent is a phosphine compound.
- the first peptide contains, at the C-terminal end, an amino acid derivative having a group represented by a formula: —CO—S—R
- R is an alkyl group having 1 to 12 carbon atoms which may be substituted by a carboxyl group, or an aryl group having 7 to 12 carbon atoms which may be substituted by a carboxyl group; and the second peptide contains, at the N-terminal end, a serine or threonine derivative having a group represented by a formula: —R 2 —S—Y 1 wherein Y 1 is a protecting group of a thiol auxiliary group, and R 2 is a methylene group which may be substituted.
- Another aspect of the present invention provides a compound represented by the following formula 1:
- Y 1 is a protecting group of a thiol auxiliary group
- Y 2 is a protecting group of an amino group
- R 1 is a hydrogen atom or a methyl group
- R 2 is a methylene group which may be substituted
- n is an integer of 1 to 3.
- Y 1 is a t-butyl group
- Y 2 is a 9-fluorenylmethoxycarbonyl group
- R 1 is a hydrogen atom or a methyl group
- R 2 is a methylene group
- n is 2.
- the thiol auxiliary group involved in the ligation spontaneously decomposes after the ligation reaction have proceeded, and therefore, a reaction step for removing the thiol auxiliary group is not particularly necessary. Therefore, the method for producing a peptide of the invention has a lower possibility that the side reaction occurs, and shows excellent ligation efficiency, in comparison with the conventional native chemical ligation methods utilizing the thiol auxiliary group.
- This usefulness of the method for producing peptide of the invention leads to an industrially highly practical method for producing a peptide in which a peptide having high stability can be easily produced in a large-scale.
- FIG. 1 shows an outline of a native chemical ligation method
- FIG. 2 shows examples of thiol auxiliary groups, which have hitherto been used in ligation
- FIG. 3 shows an outline of a method for producing a peptide of the present invention, wherein (A) shows an assumed ligation reaction route via an intermediate A in the method for producing a peptide of the invention, and (B) is a view showing ligation of a thioester group at a C-terminal end of a peptide in an N-terminal side, and a thiol auxiliary group at an N-terminal end of another peptide in a C-terminal side, in the method for producing a peptide of the invention;
- FIG. 4 shows a synthesis route of a serine or threonine derivative having a thiol auxiliary group
- FIG. 5 shows a synthesis route of a second peptide containing a serine or threonine derivative having a thiol auxiliary group at the terminal end;
- FIG. 6 shows the results of HPLC which have confirmed a synthesis of peptide 17 in Example 1 (6), wherein the chart shows the HPLC results obtained after a 24-hour ligation under condition 4 in Table 1, calculation values being as follows: C-terminal side chain having no thiol auxiliary group: 1427.78
- ligated product 1997.97 ligated product in which two N-terminal ends are ligated to a C-terminal end: 2568.16;
- FIG. 7 shows a synthesis route of a first peptide containing an amino acid derivative having a thioester group at the C-terminal end.
- a first peptide containing an amino acid derivative having a thioester group at the C-terminal end and a second peptide containing a serine or threonine derivative having a thiol auxiliary group at the N-terminal end are caused to react in the presence of a reducing agent to thereby form a intermolecular thioester between the thioester group in the amino acid derivative in the first peptide and the thiol auxiliary group in the serine or threonine derivative in the second peptide; then an amido bond is formed through an intramolecular transfer; next the thiol auxiliary group is removed; and finally a ligated product of the first peptide and the second peptide is obtained ( FIG. 3 ).
- R is a hydrogen atom or a methyl group was obtained.
- glycopeptide When the glycopeptide is synthesized, first, a synthesis of a C-terminal-side peptide containing, at the N-terminal end, a serine derivative having a thiol auxiliary group, and an N-terminal-side peptide containing, at the C-terminal end, an amino acid derivative having a thioester group was attempted, which are two part of the glycopeptide chain described above divided between the Gly residue at the 6-position and the Ser residue at the 7-position.
- the peptide chain length having the C-terminal-side peptide was extended by using an automatic synthesizer according to an Fmoc method, using an Fmoc-Leu-CLEAR Acid resin as a starting material.
- compound 12 having the following formula 12:
- Peptide 14 (pGlu-Ser-Glu-Glu-Gly-Gly-SC 6 H 4 CH 2 COOH), a peptide containing, at the C-terminal end, an amino acid derivative having a thioester group, was synthesized with reference to a method described in WO 2008/044628 ( FIG. 7 ).
- a reaction liquid is 10-fold diluted with DMF 10 minutes after the reaction. a) In each case, the reaction was performed at pH 7.0 for 24 hours. b) The yield is obtained by dividing a peak area of a desired product at 280 nm by the total of the peak area of desired product, a peak area of an unreacted C-terminal segment, and a peak area of a by-product derived from the C-terminal end. c) A reaction liquid is 10-fold diluted with DMF 10 minutes after the reaction.
- ligation was attempted by dissolving peptides 13 and 14 in 0.1 M phosphate buffer solution (pH 7.0) in a molar ratio of 1:1, and adding 0.2 M mercaptophenyl acetic acid (MPAA) thereto (condition 1 in Table 1). It was expected that the mercaptophenyl acetic acid would inhibit the hydrolysis of the thioester bond in peptide 14, and would have an action of reducing the thiol auxiliary group in peptide 13 having been protected with the disulfide bond, to start the ligation, under this condition.
- MPAA mercaptophenyl acetic acid
- glycopeptide 17 (pGlu-Ser-Glu-Glu-Gly-Gly-Ser-Asn-Ala-Tlu(GalNAcBn)-Lys-Lys-Pro-Tyr-Ile-Leu-OH) was 25%. Therefore, in order to inhibit the hydrolysis after the thioester bond is formed between the peptides, the ligation was performed under a condition in which 50% of acetonitrile was added, as shown in the condition 3 in Table 1, whereby the yield was improved up to 39%.
- the intramolecular aminolysis was performed preferentially under condition 4 in which the reaction solution was 10-fold diluted with dimethyl formamide 10 minutes after the reaction, whereby the yield was improved up to 49% ( FIG. 6 ).
- the method for producing a peptide of the present invention does not have the concern for a side reaction during a removal reaction after the ligation, because such a removal reaction is not necessary, and is a highly practical method capable of performing efficient ligation.
- the method for producing a peptide of the invention is a method in which further improvement of ligation yield can be expected by using the thiol auxiliary group having high stability.
- the method for producing a peptide of the invention contains the step of causing the first peptide to react with the second peptide in the presence of the reducing agent to give the ligated product of the first peptide and the second peptide (hereinafter sometimes may be referred to as a “ligation step”).
- the ligation step will be explained below.
- the first peptide that is a starting material in the ligation step has an amino acid derivative with a thioester group at the C-terminal end.
- the second peptide that is another starting material in the ligation step has a serine derivative with a thiol auxiliary group or a threonine derivative with a thiol auxiliary group at the N-terminal end.
- the thiol auxiliary group in the serine or threonine derivative in the second peptide reacts with the thioester group in the amino acid derivative in the first peptide in the presence of a reducing agent to form a thioester condensate that is an intermediate.
- a thioester condensate that is an intermediate.
- an S ⁇ N intramolecular transition spontaneously occurs, followed by the removal of the thiol auxiliary group.
- the thiol auxiliary group in the serine or threonine derivative, which is present at the N-terminal end of the second peptide is not particularly limited, as long as it can react with the thioester group in the amino acid derivative, which is present at the C-terminal end of the first peptide.
- groups: Y 1 —(S) n —R 2 — wherein Y 1 is a protecting group of the thiol auxiliary group, R 2 is a methylene group, which may be substituted, and n is an integer of 1 to 3 are preferable, and groups: Y 1 —(S) 2 —R 2 — wherein Y 1 and R 2 are as defined above are more preferable.
- the group R 2 is preferably a methylene group, more preferably a methylene group in which the hydrogen atom is substituted by another atom or group, and further more preferably a methylene group in which the hydrogen atom is substituted by an electron-withdrawing atom or group.
- R 2 include —CH 2 —, —CH(CF 3 )—, —CH(F)—, and the like.
- the thioester group in the amino acid derivative, which is present at the C-terminal end of the first peptide is not particularly limited as long as it is caused to react with the thiol auxiliary group in the serine or threonine derivative, which is present at the N-terminal end of the second peptide.
- groups: —CO—S—R wherein R is an alkyl group having 1 to 12 carbon atoms, which may be substituted by a carboxyl group, or an aryl group having 7 to 12 carbon atoms, which may be substituted by a carboxyl group are preferable.
- the alkyl group having 1 to 12 carbon atoms in R include a methyl group, an ethyl group, a propyl group, an isopropyl group, a butyl group, an isobutyl group, a pentyl group, an isopentyl group, a hexyl group, a heptyl group, and the like.
- Examples of the aryl group having 7 to 12 carbon atoms include a phenyl group, a benzyl group, a tolyl group, a xylyl group, a mesityl group, a cumenyl group, a phenethyl group, a naphthyl group, and the like.
- the hydrogen atom in the alkyl group having 1 to 12 carbon atoms and the aryl group having 7 to 12 carbon atoms in R described above may be substituted by a carboxyl group.
- a preferable example of R is —C 6 H 4 CH 2 COOH.
- the serine or threonine derivative is introduced into the peptide chain in the form of a compound, for example, represented by the following formula 1:
- Y 1 is a protecting group of the thiol auxiliary group
- Y 2 is a protecting group of the amino group
- R 1 is a hydrogen atom or a methyl group
- R 2 is a methylene group which may be substituted
- n is an integer of 1 to 3.
- Examples of Y 1 that is the protecting group of the thiol auxiliary group and Y 2 that is the protecting group of the amino group in the serine or threonine derivative each include a t-butyl group, a 9-fluorenylmethoxycarbonyl group, a trityl group, an acetamidomethyl group, a benzyl group, a 4-methylbenzyl group, a 4-methoxybenzyl group, a 3-nitro-2-pyridinesulfenyl group, an ethylmercapto group, a tert-butylmercapto group, and the like, Y 1 being different from Y 2 .
- Y 1 is preferably t-butyl group
- Y 2 is preferably 9-fluorenylmethoxycarbonyl group.
- R 2 is preferably a methylene group, more preferably a methylene group in which the hydrogen atom is substituted by another atom or group, and further more preferably a methylene group in which the hydrogen atom is substituted by an electron-withdrawing atom or group.
- R 2 include —CH 2 —, —CH(CF 3 )—, —CH(F)—, and the like.
- substituted methylene groups such as —CH(F)— and —CH(CF 3 )— are preferable.
- n showing the number of sulfur atoms is preferably within a range of an integer of 1 to 3, more preferably 1 or 2, and further more preferably 2.
- serine or threonine in which the amino group is protected with Fmoc and the carboxyl group is protected with Bu t is caused to react with a sulfur-containing compound such as dimethyl sulfoxide (DMSO), methyl ethyl sulfoxide or diethyl sulfoxide, and Ac 2 O and AcOH, whereby the side-chain hydroxyl group is alkylthioalkylated.
- DMSO dimethyl sulfoxide
- methyl ethyl sulfoxide or diethyl sulfoxide and Ac 2 O and AcOH
- the reaction can be performed at an appropriate temperature (for example, 10 to 60° C.) for a predetermined time.
- the alkylthioalkylated product can be recovered as, for example, a precipitate.
- the resulting precipitate can also be dissolved in an appropriate solvent such as ethyl acetate, followed by sequential washing with an aqueous saturated sodium bicarbonate solution and an aqueous saturated sodium chloride solution, and the like.
- the resulting alkylthioalkylated product is subjected to appropriate treatments such as drying and concentration, followed by separation and purification.
- the separated and purified alkylthioalkylated product is dissolved in a basic solvent containing a neutralizing agent, preferably N,N-diisopropylethylamine (DIEA) at an appropriate temperature (for example, ⁇ 10 to 10° C.) for a predetermined time to neutralize it, then the resultant product is mixed with a solvent containing a halogenating agent, preferably sulfuryl chloride at an appropriate temperature (for example, 10 to 60° C.) for a predetermined time to halogenate the alkylthioalkyl group, and after that the resulting product is mixed with a solvent containing an agent that converts to a thiol group, preferably potassium thiotosylate at an appropriate temperature (for example, 10 to 60° C.) for a predetermined time to substitute thiol for the halogen.
- a neutralizing agent preferably N,N-diisopropylethylamine (DIEA)
- DIEA N,
- a converted trisulfide can be obtained from the alkylthioalkylated product in the same manner as above.
- the converted disulfide is dissolved in TFA, preferably TFA containing thioanisole at an appropriate temperature (for example, 10 to 60° C.) for a predetermined time.
- TFA preferably TFA containing thioanisole
- the thioanisole in the TFA can trap Bu t cations, which may be derived from a decomposition of Bu t ester.
- the obtained serine or threonine derivative is subjected to proper procedures such as drying and concentration, followed by separation and purification. For the separation and purification, it is preferable to use, for example, a silica-gel chromatography.
- the second peptide can be synthesized by, for example, bonding the serine or threonine derivative to the N-terminal end of the peptide chain which has been extended by using a peptide automatic synthesizer.
- a peptide automatic synthesizer for example, an amino acid residue bonded to a resin is used as a starting material, and the peptide chain length is extended using an automatic synthesizer according to an Fmoc method.
- the serine or threonine derivative for example, Fmoc-Ser(CH 2 —S—S-Bu t )—OH
- the terminal Fmoc in the obtained resin is removed, dried it, a mixed liquid of TFA:phenol:distilled water:thioanisole is added thereto, and the mixture is stirred.
- the liquid is removed by passing nitrogen gas, to which diethyl ether is added to precipitate a peptide.
- the peptide is extracted with an aqueous acetonitrile solution, and the resin is filtered. Purification with a reversed phase HPLC can provide a second peptide.
- the synthesis reaction of the second peptide may be performed at a proper temperature (for example, 10 to 60° C.) for a predetermined time.
- an amino acid residue modified with sugar or the like may be introduced in a conventional technique.
- a resin whose peptide chain length is extended, and a sugar-modified amino acid protected with Fmoc are added to a solution of HBTU-HOBt/DMF and DIEA in N-methylpyrrolidone (NMP), and the reaction is performed at a proper temperature (for example, 50° C.) for a predetermined time, whereby the sugar-modified amino acid residue can be added on the peptide chain.
- NMP N-methylpyrrolidone
- Examples of the thus obtained second peptide may include peptide 13 described above.
- the synthesis of the first peptide is not particularly limited as long as a peptide containing, at the C-terminal end, an amino acid derivative having a thioester group can be obtained.
- the synthesis can be performed according to, for example, a direct synthesis method using a Boc method (Hojo, H.; Aimoto, S. Bull. Chem. Soc. Jpn., 64, 111-117, (1991)); a direct synthesis method using an Fmoc method (Li, X.; Kawakami, T.; Aimoto, S.
- a peptide containing, at the C-terminal side, an amino acid derivative having a thioester group can be synthesized through following steps (i) to (iv).
- step (i) the deprotection of the Fmoc group is performed by, for example, treating an Fmoc-CLEAR amide resin with piperidine/N-methylpyrrolidone (NMP), and the resulting resin is caused to react with Fmoc-N(R)—CH(CH 2 SY)—C( ⁇ O)OH in HOBt/NMP and N,N′-dicyclohexyl carbodiimide (DCC)/NMP.
- the reaction may be performed at an appropriate temperature (for example, 40 to 60° C.).
- the group R in Fmoc-N(R)—CH(CH 2 SY)—C( ⁇ O)OH is an alkyl group having 1 to 12 carbon atoms or an aralkyl group having 7 to 12 carbon atoms.
- the alkyl group can include a methyl group, an ethyl group, a propyl group, a butyl group, a pentyl group, a hexyl group, and the like, which may be linear, branched or cyclic hydrocarbon groups.
- Examples of the aralkyl group can include a benzyl group, and the like.
- R is preferably a methyl group, an ethyl group, an isobutyl group or a benzyl group.
- Step (ii) is a step in which after the deprotection of the Fmoc group in the Fmoc-(amino acid residue) n -N(R)—CH(CH 2 SY)—C( ⁇ O)NH-resin in which n is 0 or 1, obtained in step (i), is performed, the resulting product is caused to react with an Fmoc-amino acid, if necessary, to produce an Fmoc-(amino acid residue) n -N(R)—CH(CH 2 SY)—C( ⁇ O)NH-resin in which n is 1 or 2, and then the deprotection of the Fmoc group and the reaction with the Fmoc-amino acid are repeated to produce an X—N(R)—CH(CH 2 SY)—C( ⁇ O)—NH-resin.
- step (i) the Fmoc-(amino acid residue) n -N(R)—CH(CH 2 SY)—C( ⁇ O)NH-resin is produced by causing the Fmoc-(amino acid residue) n -N(R)—CH(CH 2 SY)—C( ⁇ O)OH (n is 1) to react, the Fmoc-amino acid may be caused to react or not in step (ii).
- step (ii) specifically, the reaction product from step (i) is washed with NMP, and then the resin is shaken in Ac 2 O-DIEA/NMP for 5 minutes. After washing with NMP and the removal of the Fmoc group with piperidine/NMP are performed, a solution of Fmoc-Gly and hexafluorophosphate, O-(7-azabenzotriazole-1-yl)-N,N,N′,N′-tetramethyluronium (HATU), and N,N-diisopropylethylamine (DIEA) dissolved in NMP is added thereto, and the reaction is performed.
- HATU O-(7-azabenzotriazole-1-yl)-N,N,N′,N′-tetramethyluronium
- DIEA N,N-diisopropylethylamine
- the reaction may be performed at an appropriate temperature (for example, 40 to 60° C.) for a predetermined time.
- an appropriate temperature for example, 40 to 60° C.
- the dehydration of the Fmoc group and reaction with the Fmoc-amino acid are repeated by using a conventional peptide synthesizer in accordance with, for example, a Fast Moc method, whereby the peptide chain length can be extended.
- Step (iii) is a step in which the X—N(R)—CH(CH 2 SY)—C( ⁇ O)—NH-resin obtained in step (ii) is caused to react with a deprotecting agent (for example, trifluoroacetic acid, and the like), whereby the elimination from the resin and deprotection of the thiol group are performed.
- a deprotecting agent for example, trifluoroacetic acid, and the like
- Reagent K is added to the product (resin) in step (ii), and the reaction can be performed at room temperature.
- Step (iv) is a step in which the compound obtained in step (iii) is caused to react with an acidic thiol to produce a thioester compound.
- the acidic thiol to be used here may include mercaptocarboxylic acid and a mixture of mercaptan and a carboxylic acid.
- HSCH 2 CH 2 COOH(MPA), HSC 6 H 4 CH 2 COOH(MPAA), or a mixture of thiophenol and acetic acid can be more preferably used.
- step (iv) TFA is removed from the product from step (iii) through nitrogen stream, to which an ether is added to generate a precipitate.
- the precipitate is washed with an ether, and then is dried.
- the crude peptide is extracted with an aqueous acetonitrile solution containing TFA, which is diluted with an aqueous solution of acetonitrile of acidic thiol such as an aqueous solution of 3-mercaptopropionic acid or 4-mercaptophenylacetic acid, and the resulting product is allowed to stand for several hours to several ten hours, whereby a peptide thioester compound represented by X—S—CH 2 CH 2 COOH or X—S—C 6 H 4 —CH 2 COOH can be obtained.
- Examples of the first peptide obtained through steps (i) to (iv) can include peptide 14 described above.
- a compound represented by Z—N(R)—CH(CH 2 SY)—C( ⁇ O)OH wherein Z is a hydrogen atom or a 9-fluorenylmethoxycarbonyl group, R is an alkyl group having 1 to 12 carbon atoms or an aralkyl group having 7 to 12 carbon atoms, and Y is a protecting group of thiol, R and Y being described in detail above, is synthesized as follows:
- a compound represented by YSCH 2 CH(NH 2 )C( ⁇ O)OH wherein Y is a protecting group of thiol group or cysteine in which the thiol group is protected and a compound represented by R 1 CHO wherein R 1 is a hydrogen atom, an alkyl group having 1 to 11 carbon atoms or an aryl group having 6 to 11 carbon atoms are caused to react to produce a compound represented by YSCH 2 CH(N ⁇ CHR 1 )C( ⁇ O)OH wherein Y and R 1 are as defined above.
- the alkyl group in R 1 is preferably an alkyl group having 1 to 9 carbon atoms, more preferably an alkyl group having 1 to 6 carbon atoms.
- Examples thereof may include a methyl group, an ethyl group, a propyl group, a butyl group, a pentyl group, a hexyl group, and the like, which may be any of linear, branched or cyclic hydrocarbons.
- Particularly preferable R 1 s are a methyl group, an isopropyl group and a phenyl group.
- the reaction thereof with the compound represented by R 1 CHO can be performed by dissolving the cysteine in which the thiol group is protected in water containing ethanol and potassium hydroxide, adding the compound represented by R 1 CHO thereto, and stirring the mixture at an appropriate temperature (for example, room temperature) for a predetermined time.
- the compound represented by YSCH 2 CH(N ⁇ CHR 1 )C( ⁇ O)OH wherein Y and R 1 are as defined above is caused to react with a hydrogenating agent (for example, NaBH 4 or NaBH 3 CN) to produce a compound represented ,by YSCH 2 CH(NHCH 2 R 1 )C( ⁇ O)OH wherein Y and R 1 are as defined above, and, if necessary, the resulting product is caused to react with 9-fluorenylmethoxycarbonyl-N-hydroxysuccinimide ester (hereinafter “Fmoc-OSu”) to protect the amino group with Fmoc group, whereby a compound represented by Z—N(R)—CH(CH 2 SY)—C( ⁇ O)OH wherein Z is a hydrogen atom or a 9-fluorenylmethoxycarbonyl group, R is a alkyl group having 1 to 12 carbon atoms, or an aralkyl group having 7 to 12 carbon atom
- the hydrogenating agent may be caused to react in the presence of a base such as sodium hydroxide.
- a base such as sodium hydroxide.
- the hydrogenating agent for example, NaBH 4 or NaBH 3 CN
- the compound represented by YSCH 2 CH(N ⁇ CHR 1 )C( ⁇ O)OH is added to the compound represented by YSCH 2 CH(N ⁇ CHR 1 )C( ⁇ O)OH, and the mixture is stirred at a proper temperature, whereby the reaction can be performed.
- a base is not particularly necessary.
- the amino group when the amino group is protected with the Fmoc group by causing it to react with Fmoc-OSu, the amino group can be protected with the Fmoc group by causing it to react with the Fmoc group in the presence of sodium carbonate in a proper solvent (for example, 1,2-dimethoxyethane).
- a proper solvent for example, 1,2-dimethoxyethane
- the ligation reaction of the first peptide and the second peptide can be performed in the presence of a reducing agent in a buffer solution or a mixed solution of a buffer and an organic solvent.
- the reducing agent is not particularly limited, as long as it can reduce the thiol auxiliary group in the serine or threonine derivative to a state having a free thiol group. It is preferable to use, for example, a water-soluble phosphine such as triscarboxyethyl phosphine (TCEP) or trishydroxymethyl phosphine, trimethyl phosphine, triethyl phosphine, triisopropyl phosphine, tributyl phosphine, triphenyl phosphine, or the like; it is more preferable to use water-soluble phosphine, further more preferable to use TCEP.
- TCEP triscarboxyethyl phosphine
- TCEP trishydroxymethyl phosphine
- trimethyl phosphine triethyl phosphine
- triisopropyl phosphine tributyl phos
- the concentration of the reducing agent can be suitably adjusted depending to the kind of the reducing agent, as long as it can reduce the thiol auxiliary group in the serine or threonine derivative to a state having the free thiol group.
- concentration is preferably from 7.5 to 50 mM, more preferably from 15 to 30 mM.
- the buffer solution to be used in the ligation reaction of the first peptide and the second peptide can include phosphate buffer solution, Tris-hydrochloric acid buffer solution, citrate buffer solution, and the like. Among these, the phosphate buffer solution is preferable.
- the mixed solution of the buffer solution and the organic solvent used in the ligation reaction of the first peptide and the second peptide can include mixed solutions of the buffer solution described above and an organic solvent such as acetonitrile, DMF, or NMP. Among these, the mixed solution of phosphate buffer solution and acetonitrile is preferable.
- the concentrations of the buffer solution and the mixed solution of buffer solution and organic solvent, and the mixing ratio of the mixed solution can be suitably adjusted depending on the addition amounts of the first peptide used and the second peptide used, and the kinds of the buffer solution and the organic solvent.
- the concentration of the phosphate buffer solution used in the ligation reaction is preferably from 0.1 to 1.0 M, more preferably from 0.1 to 0.5 M, and further more preferably from 0.1 to 0.2 M.
- the mixing ratio between the 0.1 M phosphate buffer solution and acetonitrile in the mixed solution used in the ligation reaction is preferably 1:1 of 0.1 M phosphate buffer solution and acetonitrile.
- the ligation reaction is performed at a pH of preferably 5 to 9, more preferably 6 to 8, and further more preferably 7 ⁇ 0.2.
- the temperature and the time of the ligation reaction can be suitably adjusted.
- the reaction temperature can be set at room temperature (10 to 40° C.) and the reaction time can be set to a time during which the ligated product of the first peptide and the second peptide can be obtained in a predetermined amount.
- a conventional method used for confirming a peptide can be used without any limitation, and for example, a method using an HPLC is preferable.
- the yield of the ligated product of the first peptide and the second peptide is measured as follows:
- the ligated product of the first peptide and the second peptide is subjected to an HPLC measurement by using a C18 column, and an aqueous acetonitrile solution containing 0.1% (v/v) TFA as an eluent.
- the yield is calculated by dividing a peak area of a desired product at 280 nm by the total of the peak area of desired product, a peak area of an unreacted C-terminal segment, and a peak area of a by-product derived from the C-terminal end.
- a Lys(Boc)-Lys(Boc)-Pro-Tyr(Bu t )-Ile-Leu-CLEAR Acid resin was obtained from a starting material, Fmoc-Leu-CLEAR Acid resin (0.49 meq/g, 200 mg, 0.1 mmol) by the use of an automatic synthesizer according to a FastMoc method.
- the resulting product was condensed with Fmoc-Ala-OH (33 mg, 0.1 mmol) and then Fmoc-Asn(Trt)-OH (59.7 mg, 0.1 mmol) by using 1 M DCC (150 ⁇ l) and 1 M HOBt (150 ⁇ l). Finally, the resulting product was condensed with Fmoc-Ser(CH 2 —S—S-Bu t )—OH (31 mg, 0.066 mmol) by using 1 M DCC (99 ⁇ l) and 1 M HOBt (99 ⁇ l).
- the resin was condensed with Boc-pGlu-OH (46 mg, 0.20 mmol) using 0.45 M HBTU, HOBt/DMF (420 0.19 mmol) and DIEA (70 0.40 mmol) to obtain Boc-pGlu-Ser(Bu t )-Glu(OBu t )-Glu(OBu t )-Gly-CLEAR amide resin (330 mg).
- condition 1 in Table 1 peptide 13 and peptide 14 (20 nmol, each) were dissolved in a 0.1 M sodium phosphate buffer (pH 7.2, 4 ⁇ l) containing 6 M guanidine hydrochloride and 0.2 M MPAA, and the mixture was allowed to stand at room temperature overnight.
- condition 2 in Table 1 peptide 13 and peptide 14 (20 nmol, each) were dissolved in a 0.1 M sodium phosphate buffer (pH 7.0, 4 ⁇ l) containing 7.5 mM triscarboxyethyl phosphine (TCEP), and the mixture was allowed to stand at room temperature overnight.
- TCEP triscarboxyethyl phosphine
- condition 3 in Table 1 peptide 13 and peptide 14 (20 nmol, each) were dissolved in 50% 0.1 M sodium phosphate buffer-acetonitrile (pH 7.0, 4 ⁇ l) containing 7.5 mM triscarboxyethyl phosphine (TCEP), and the mixture was allowed to stand overnight.
- condition 4 in Table 1 peptide 13 and peptide 14 (20 nmol, each) were dissolved in 50% 0.1 M sodium phosphate buffer-acetonitrile (pH 7.0, 4 ⁇ l) containing 7.5 mM triscarboxyethyl phosphine (TCEP), and DMF was added thereto after 10 minutes, and the mixture was allowed to stand overnight.
- Fmoc-Thr-OBu t (1.3 g, 3.3 mmol) was dissolved in DMSO (6.0 ml, 85 mmol), Ac 2 O (4.0 ml, 42 mmol) and AcOH (6.0 ml, 0.10 mol), and the mixture was caused to react at room temperature for two days. After vacuum concentration, distilled water was added thereto until precipitate was generated, and a supernatant was removed therefrom. The precipitate was dissolved in ethyl acetate, the resulting solution was transferred to a separating funnel, and after that, was washed with an aqueous saturated sodium bicarbonate solution and an aqueous saturated sodium chloride solution.
- a protein or glycoprotein having high stability can be solution-phase synthesized in a high yield, while the generation of by-products is restrained.
- the protein or glycoprotein obtained by the method for producing a peptide of the invention can be utilized in a production of pharmaceutical products.
- the glycoprotein produced by the present invention can be expected to show the same activity as that of a naturally occurring glycoprotein, such as an activity of bonding to a specific cell receptor as one kind of nerve transmitter substances.
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Abstract
An object of the present invention is to provide a novel method for producing a peptide utilizing a ligation reaction in which ligation efficiency is excellent and side reactions to other functional groups in the peptide are hard to occur, in comparison with the conventional native chemical ligation methods utilizing the thiol auxiliary group. The present invention provides a method for producing a peptide which comprises a step of causing a first peptide and a second peptide to react in the presence of a reducing agent to obtain a ligated product of the first peptide and the second peptide, wherein the first peptide contains, at the C-terminal end, an amino acid derivative having a thioester group, and the second peptide contains, at the N-terminal end, a serine or threonine derivative having a thiol auxiliary group.
Description
- The present invention relates to a novel method for synthesizing a peptide. More specifically, the present invention relates to a novel method for synthesizing a peptide utilizing a serine or threonine derivative having a thiol auxiliary group, and an amino acid derivative having a thioester group.
- As a method for chemically synthesizing a peptide, a thioester method in which peptide thioester segments prepared by a solid-phase method are repeatedly condensed (Non-Patent Document 1), and a native chemical ligation method (Non-Patent Document 2) are generally used.
- The thioester method realizes a segment-to-segment condensation by activating, with silver ions, the thioester terminal groups of the partially protected peptide thioester. On the other hand, according to the native chemical ligation method, an intermolecular thioester bond is formed between a thioester terminal group of a peptide thioester having no protecting group and a cysteine residue at an N-terminal end of another peptide, followed by formation of a peptide bond by an intermolecular aminolysis reaction of the amino group of the cysteine residue (
FIG. 1 ). - The native chemical ligation method is more convenient than the thioester method because the peptide prepared by the solid-phase method can be used as it is in the condensation. Accordingly, the native chemical ligation method is becoming the mainstream of a chemical synthesis of a protein.
- The native chemical ligation method, however, has a defect in which condensation can be performed only between peptide thioester having a thioester terminal group but no protecting group and a peptide having cysteine at the N-terminal end.
- In order to overcome the defect described above, various modified thiol auxiliary groups that can be removed after ligation, have hitherto been developed by many researchers (
FIG. 2 ). The native chemical ligation methods using the thiol auxiliary group include, for example, a method in which after ligation of cysteine is performed, the resulting cysteine is converted into alanine through a desulfurization reaction (Non-Patent Documents 3-5); a method in which after ligation is performed by using a phenylalanine residue having a thiol auxiliary group at the β-potision, the conversion into phenylalanine is performed through a desulfurization reaction (Non-Patent Document 6); a method in which after ligation of homocysteine is performed, conversion into a methionine residue is performed through methylation (Non-Patent Document 7); a method in which a thiol auxiliary group is bonded to an acetamido group or hydroxyl group of a sugar chain, and the ligation is performed, as shown in 8 or 9 ofFIG. 2 (Non-Patent Documents 8-12); moreover, a method in which a thiol auxiliary group is bonded to a side-chain carboxyl group, and the ligation is performed (Non-Patent Document 13), and the like. - The conventional native chemical ligation methods utilizing the thiol auxiliary group described above, however, have various defects.
- According to the methods described in Non-Patent Documents 3-5 and 6, the peptide is adsorbed on a metal, thus resulting in the lowering of a recovery rate, because of the use of a metal catalyst in the desulfurization reaction, and, furthermore, side reactions such as demethylthiolation in the methionine residue of the peptide may occur.
- According to the method described in Non-Patent Document 7, other functional groups may also be methylated when the homocysteine residue is methylated.
- According to the methods described in Non-Patent Documents 8-12 and 13, when the acetamido group is used, the same situations as described above result because the desulfurization reaction is finally performed, and when the sugar chain hydroxyl group is used, β-elimination of the sugar chain or racemization of α-carbon may possibly occur because an alkaline treatment is finally performed for the removal.
- According to the conventional native chemical ligation methods utilizing the thiol auxiliary group, therefore, the efficiency of the ligation is low, and side reactions to the functional groups in the various amino acid residues in the peptide may possibly occur.
-
- Non-Patent Document 1: Hojo, H.; Aimoto, S. Bull. Chem. Soc. Jpn. 1991, 64, 111-117
- Non-Patent Document 2: Dawson, P. E.; Muir, T. W.; Clark-Lewis, I.; Kent, S. B. Science 1994, 266, 776-779
- Non-Patent Document 3: Yan, L. Z.; Dawson, P. E. J. Am. Chem. Soc. 2001, 123, 526-533.
- Non-Patent Document 4: Pentelute, B. L.; Kent, S. B. H. Org. Lett. 2007, 9, 687-690.
- Non-Patent Document 5: Wan, Q.; Danishefsky, S. J. Angew. Chem. Int. Ed. 2007, 46, 9248-9252.
- Non-Patent Document 6: Crich, D.; Banerjee, A. J. Am. Chem. Soc. 2007, 129, 10064-10065. (Phe) X-Met site
- Non-Patent Document 7: Saporito, A.; Marasco, D.; Chambery, A.; Botti, P.; Monti, S. M.; Pedone, C.; Ruvo, M. Biopolymers 2006, 83, 508-518.
- Non-Patent Document 8: Brik, A.; Ficht, S.; Yang, Y.-Y.; Bennett, C. S.; Wong, C.-H. J. Am. Chem. Soc. 2006, 128, 15026-15033.
- Non-Patent Document 9: Brik, A.; Yang, Y.-Y.; Ficht, S.; Wong, C.-H. J. Am. Chem. Soc. 2006, 128, 5626-5627.
- Non-Patent Document 10: Yang, Y.-Y.; Ficht, S.; Brik, A.; Wong, C.-H. J. Am. Chem. Soc. 2007, 129, 7690-7701.
- Non-Patent Document 11: Payne, R. J.; Ficht, S.; Tang, S.; Brik, A.; Yang, Y.-Y.; Case, D. A.; Wong, C.-H. J. Am. Chem. Soc. 2007, 129, 13527-13536.
- Non-Patent Document 12: Ficht, S.; Payne, R. J.; Brik, A.; Wong, C.-H. Angew. Chem. Int. Ed 2007, 46, 5975-5979.
-
- Non-Patent Document 13: Lutsky, M.-Y.; Nepomniaschiy, N.; Brik, A. Chem. Commun. 2008, 1229-1231.
- An object to be solved by the present invention is to solve the problems of the prior art described above. That is, the object to be solved by the present invention is to provide a novel method for producing a peptide utilizing a ligation reaction in which ligation efficiency is excellent and side reactions to other functional groups in the peptide are hard to occur, in comparison with the conventional native chemical ligation methods utilizing the thiol auxiliary group. Furthermore, a second object to be solved by the present invention is to provide a compound having a novel thiol auxiliary group to be used in the production method described above.
- As a result of outstanding studies for solving the problems described above, the present inventors have found that when a first peptide containing an amino acid derivative having a thioester group and a second peptide containing a serine or threonine derivative having a thiol auxiliary group are caused to react in the presence of a reducing agent, a ligated product of the first peptide and the second peptide can be obtained with high ligation efficiency and without the occurrence of side reaction to other functional groups in the peptide; and have reached the accomplishment of the present invention.
- Thus, the present invention provides a method for producing a peptide which comprises a step of causing a first peptide and a second peptide to react in the presence of a reducing agent to obtain a ligated product of the first peptide and the second peptide, wherein the first peptide contains, at the C-terminal end, an amino acid derivative having a thioester group, and the second peptide contains, at the N-terminal end, a serine or threonine derivative having a thiol auxiliary group.
- According to a preferred embodiment of the production method of the present invention, the thiol auxiliary group is a group containing a disulfide bond.
- According to a preferred embodiment of the production method of the present invention, the reducing agent is a phosphine compound.
- According to a preferred embodiment of the production method of the present invention, the first peptide contains, at the C-terminal end, an amino acid derivative having a group represented by a formula: —CO—S—R
- wherein R is an alkyl group having 1 to 12 carbon atoms which may be substituted by a carboxyl group, or an aryl group having 7 to 12 carbon atoms which may be substituted by a carboxyl group; and the second peptide contains, at the N-terminal end, a serine or threonine derivative having a group represented by a formula: —R2—S—Y1 wherein Y1 is a protecting group of a thiol auxiliary group, and R2 is a methylene group which may be substituted.
- Another aspect of the present invention provides a compound represented by the following formula 1:
- wherein Y1 is a protecting group of a thiol auxiliary group, Y2 is a protecting group of an amino group, R1 is a hydrogen atom or a methyl group, R2 is a methylene group which may be substituted, and n is an integer of 1 to 3.
- According to a preferred embodiment of the compound of the present invention, Y1 is a t-butyl group, Y2 is a 9-fluorenylmethoxycarbonyl group, R1 is a hydrogen atom or a methyl group, R2 is a methylene group, and n is 2.
- According to the method for producing a peptide of the present invention, the thiol auxiliary group involved in the ligation spontaneously decomposes after the ligation reaction have proceeded, and therefore, a reaction step for removing the thiol auxiliary group is not particularly necessary. Therefore, the method for producing a peptide of the invention has a lower possibility that the side reaction occurs, and shows excellent ligation efficiency, in comparison with the conventional native chemical ligation methods utilizing the thiol auxiliary group. This usefulness of the method for producing peptide of the invention leads to an industrially highly practical method for producing a peptide in which a peptide having high stability can be easily produced in a large-scale.
-
FIG. 1 shows an outline of a native chemical ligation method; -
FIG. 2 shows examples of thiol auxiliary groups, which have hitherto been used in ligation; -
FIG. 3 shows an outline of a method for producing a peptide of the present invention, wherein (A) shows an assumed ligation reaction route via an intermediate A in the method for producing a peptide of the invention, and (B) is a view showing ligation of a thioester group at a C-terminal end of a peptide in an N-terminal side, and a thiol auxiliary group at an N-terminal end of another peptide in a C-terminal side, in the method for producing a peptide of the invention; -
FIG. 4 shows a synthesis route of a serine or threonine derivative having a thiol auxiliary group; -
FIG. 5 shows a synthesis route of a second peptide containing a serine or threonine derivative having a thiol auxiliary group at the terminal end; -
FIG. 6 shows the results of HPLC which have confirmed a synthesis of peptide 17 in Example 1 (6), wherein the chart shows the HPLC results obtained after a 24-hour ligation undercondition 4 in Table 1, calculation values being as follows: C-terminal side chain having no thiol auxiliary group: 1427.78 - ligated product: 1997.97
ligated product in which two N-terminal ends are ligated to a C-terminal end: 2568.16; and -
FIG. 7 shows a synthesis route of a first peptide containing an amino acid derivative having a thioester group at the C-terminal end. - The present invention will be more specifically described below. According to the method of the invention, a first peptide containing an amino acid derivative having a thioester group at the C-terminal end and a second peptide containing a serine or threonine derivative having a thiol auxiliary group at the N-terminal end are caused to react in the presence of a reducing agent to thereby form a intermolecular thioester between the thioester group in the amino acid derivative in the first peptide and the thiol auxiliary group in the serine or threonine derivative in the second peptide; then an amido bond is formed through an intramolecular transfer; next the thiol auxiliary group is removed; and finally a ligated product of the first peptide and the second peptide is obtained (
FIG. 3 ). - The present invention will be explained through a synthesis of a glycopeptide shown in Example described in the present specification, below. First, two kinds of amino acid derivatives (a serine derivative and a threonine derivative) having a thiol auxiliary group were synthesized according to a method described in
FIG. 4 . By the use of serine or threonine wherein an amino group was protected with a 9-fluorenylmethoxycarbonyl group (Fmoc) and a carboxyl group was protected with a t-butyl group (But) as a starting material, its side-chain hydroxyl group was methylthiomethylated with dimethyl sulfoxide (DMSO)/Ac2O/AcOH, and then the resulting product was caused to react with potassium thiotosylate (TsSK) and t-butyl mercaptan to convert it into a disulfide. Then, the resulting converted disulfide was treated with TFA to remove the But ester, whereby a serine or threonine derivative having the following formula 2: - wherein R is a hydrogen atom or a methyl group was obtained.
- By utilizing the serine derivative or threonine derivative obtained as above, a synthesis of a glycopeptide contulakin-G (pGlu-Ser-Glu-Glu-Gly-Gly-Ser-Asn-Ala-Thr(GalNAc)-Lys-Lys-Pro-Tyr-Ile-Leu-OH) was attempted. Meanwhile, although a natural contulakin-G has Ga1-GaINAc as a sugar chain which bonds to Thr residue at the 10-position, a synthesis of a contulakin-G having a monosaccharide which bonds to Thr residue at the 10-position was attempted here, in order to confirm the progress of the reaction according to the present invention.
- When the glycopeptide is synthesized, first, a synthesis of a C-terminal-side peptide containing, at the N-terminal end, a serine derivative having a thiol auxiliary group, and an N-terminal-side peptide containing, at the C-terminal end, an amino acid derivative having a thioester group was attempted, which are two part of the glycopeptide chain described above divided between the Gly residue at the 6-position and the Ser residue at the 7-position.
- According to a method described in
FIG. 5 , the peptide chain length having the C-terminal-side peptide was extended by using an automatic synthesizer according to an Fmoc method, using an Fmoc-Leu-CLEAR Acid resin as a starting material. Into the extended peptide chain was introduced compound 12 having the following formula 12: - which was synthesized by using HBTU (2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate)-HOBt (Hydroxybenzotriazole) according to a conventional method, and then the peptide chain length was further extended, into which the serine derivative having the thiol auxiliary group was introduced by utilizing an N,N′-dicyclohexylcarbodiimide (DCC)—HOBt method. The resulting peptide chain was treated with TFA, and the peptide was eliminated from the resin, followed by deprotection. Purification by using a reversed phase HPLC was made to obtain peptide 13 having, at the N-terminal end, the serine derivative having the thiol auxiliary group, represented by the following formula 13:
- Peptide 14 (pGlu-Ser-Glu-Glu-Gly-Gly-SC6H4CH2COOH), a peptide containing, at the C-terminal end, an amino acid derivative having a thioester group, was synthesized with reference to a method described in WO 2008/044628 (
FIG. 7 ). - The two kind peptides,
peptide 13 and peptide 14, obtained as above, were condensed according to a ligation method under various conditions shown in Table 1. -
TABLE 1 Reaction Condition a) Yield b) 1: 0.1M phosphate buffer solution 0% (including 0.2M MPAA) 2: 0.1M phosphate buffer solution 25% (including 7.5 mM TCEP) 3: 0.2M phosphate buffer solution 39% (including 15 mM TCEP): CH3CN (1:1) 4: 0.1M phosphate buffer solution 49% (including 15 mM TCEP): CH3CN (1:1) c) a) In each case, the reaction was performed at pH 7.0 for 24 hours. b) The yield is obtained by dividing a peak area of a desired product at 280 nm by the total of the peak area of desired product, a peak area of an unreacted C-terminal segment, and a peak area of a by-product derived from the C-terminal end. c) A reaction liquid is 10-fold diluted with DMF 10 minutes after the reaction.
a) In each case, the reaction was performed at pH 7.0 for 24 hours. b) The yield is obtained by dividing a peak area of a desired product at 280 nm by the total of the peak area of desired product, a peak area of an unreacted C-terminal segment, and a peak area of a by-product derived from the C-terminal end. c) A reaction liquid is 10-fold diluted withDMF 10 minutes after the reaction. - First, ligation was attempted by dissolving
peptides 13 and 14 in 0.1 M phosphate buffer solution (pH 7.0) in a molar ratio of 1:1, and adding 0.2 M mercaptophenyl acetic acid (MPAA) thereto (condition 1 in Table 1). It was expected that the mercaptophenyl acetic acid would inhibit the hydrolysis of the thioester bond in peptide 14, and would have an action of reducing the thiol auxiliary group inpeptide 13 having been protected with the disulfide bond, to start the ligation, under this condition. However, the ligation did not occur at all, and only the C-terminal peptide from which the thiol auxiliary group had been removed and the N-terminal thioester, and their hydrolyzates were able to be obtained. From this result, it was expected that the thiol auxiliary group quickly decomposed just after it was reduced to a free thiol group, and that the intramolecular aminolysis, which occurred after the thioester bond was formed between the two peptides, proceeded very slowly in comparison with a case of utilizing Cys residue, and a thioester exchange occurred quickly with the mercaptophenyl acetic acid added. Therefore, in order to inhibit the tioester exchange with the thiol added, the ligation was attempted in the absence of the thiol underconditions 2 to 4 subsequently described. In addition, in order to reduce the thiol auxiliary group into a state in which a free thiol group was present, the addition of a reducing agent, tris(2-carboxyethyl)phosphine (TCEP) to the solution was attempted. First, when the ligation was performed undercondition 2 in which only phosphate buffer including TCEP is used, the yield of glycopeptide 17 (pGlu-Ser-Glu-Glu-Gly-Gly-Ser-Asn-Ala-Tlu(GalNAcBn)-Lys-Lys-Pro-Tyr-Ile-Leu-OH) was 25%. Therefore, in order to inhibit the hydrolysis after the thioester bond is formed between the peptides, the ligation was performed under a condition in which 50% of acetonitrile was added, as shown in thecondition 3 in Table 1, whereby the yield was improved up to 39%. Further, since the intermolecular thioester was formed quickly in about 10 minutes, the intramolecular aminolysis was performed preferentially undercondition 4 in which the reaction solution was 10-fold diluted withdimethyl formamide 10 minutes after the reaction, whereby the yield was improved up to 49% (FIG. 6 ). - From the results described above, it was found that the method for producing a peptide of the present invention does not have the concern for a side reaction during a removal reaction after the ligation, because such a removal reaction is not necessary, and is a highly practical method capable of performing efficient ligation. In addition, the method for producing a peptide of the invention is a method in which further improvement of ligation yield can be expected by using the thiol auxiliary group having high stability.
- The method for producing a peptide of the invention contains the step of causing the first peptide to react with the second peptide in the presence of the reducing agent to give the ligated product of the first peptide and the second peptide (hereinafter sometimes may be referred to as a “ligation step”). The ligation step will be explained below.
- The first peptide that is a starting material in the ligation step has an amino acid derivative with a thioester group at the C-terminal end. The second peptide that is another starting material in the ligation step has a serine derivative with a thiol auxiliary group or a threonine derivative with a thiol auxiliary group at the N-terminal end.
- The thiol auxiliary group in the serine or threonine derivative in the second peptide reacts with the thioester group in the amino acid derivative in the first peptide in the presence of a reducing agent to form a thioester condensate that is an intermediate. In the formed thioester condensate, an S→N intramolecular transition spontaneously occurs, followed by the removal of the thiol auxiliary group. Accordingly, the thiol auxiliary group in the serine or threonine derivative, which is present at the N-terminal end of the second peptide, is not particularly limited, as long as it can react with the thioester group in the amino acid derivative, which is present at the C-terminal end of the first peptide. For example, groups: Y1—(S)n—R2— wherein Y1 is a protecting group of the thiol auxiliary group, R2 is a methylene group, which may be substituted, and n is an integer of 1 to 3 are preferable, and groups: Y1—(S)2—R2— wherein Y1 and R2 are as defined above are more preferable. The group R2 is preferably a methylene group, more preferably a methylene group in which the hydrogen atom is substituted by another atom or group, and further more preferably a methylene group in which the hydrogen atom is substituted by an electron-withdrawing atom or group. Examples of R2 include —CH2—, —CH(CF3)—, —CH(F)—, and the like. The thioester group in the amino acid derivative, which is present at the C-terminal end of the first peptide, is not particularly limited as long as it is caused to react with the thiol auxiliary group in the serine or threonine derivative, which is present at the N-terminal end of the second peptide. For example, groups: —CO—S—R wherein R is an alkyl group having 1 to 12 carbon atoms, which may be substituted by a carboxyl group, or an aryl group having 7 to 12 carbon atoms, which may be substituted by a carboxyl group are preferable. Examples of the alkyl group having 1 to 12 carbon atoms in R include a methyl group, an ethyl group, a propyl group, an isopropyl group, a butyl group, an isobutyl group, a pentyl group, an isopentyl group, a hexyl group, a heptyl group, and the like. Examples of the aryl group having 7 to 12 carbon atoms include a phenyl group, a benzyl group, a tolyl group, a xylyl group, a mesityl group, a cumenyl group, a phenethyl group, a naphthyl group, and the like. The hydrogen atom in the alkyl group having 1 to 12 carbon atoms and the aryl group having 7 to 12 carbon atoms in R described above may be substituted by a carboxyl group. A preferable example of R is —C6H4CH2COOH.
- The serine or threonine derivative is introduced into the peptide chain in the form of a compound, for example, represented by the following formula 1:
- wherein Y1 is a protecting group of the thiol auxiliary group, Y2 is a protecting group of the amino group, R1 is a hydrogen atom or a methyl group, R2 is a methylene group which may be substituted, and n is an integer of 1 to 3.
- Examples of Y1 that is the protecting group of the thiol auxiliary group and Y2 that is the protecting group of the amino group in the serine or threonine derivative each include a t-butyl group, a 9-fluorenylmethoxycarbonyl group, a trityl group, an acetamidomethyl group, a benzyl group, a 4-methylbenzyl group, a 4-methoxybenzyl group, a 3-nitro-2-pyridinesulfenyl group, an ethylmercapto group, a tert-butylmercapto group, and the like, Y1 being different from Y2. Y1 is preferably t-butyl group, and Y2 is preferably 9-fluorenylmethoxycarbonyl group.
- In the serine or threonine derivative, R2 is preferably a methylene group, more preferably a methylene group in which the hydrogen atom is substituted by another atom or group, and further more preferably a methylene group in which the hydrogen atom is substituted by an electron-withdrawing atom or group. Examples of R2 include —CH2—, —CH(CF3)—, —CH(F)—, and the like. In order to improve the stability of the thiol auxiliary group and increase the ligation yield, substituted methylene groups such as —CH(F)— and —CH(CF3)— are preferable.
- In the serine or threonine derivative, n showing the number of sulfur atoms is preferably within a range of an integer of 1 to 3, more preferably 1 or 2, and further more preferably 2.
- The synthesis of the serine or threonine derivative is shown below (
FIG. 8 ) - First, serine or threonine in which the amino group is protected with Fmoc and the carboxyl group is protected with But is caused to react with a sulfur-containing compound such as dimethyl sulfoxide (DMSO), methyl ethyl sulfoxide or diethyl sulfoxide, and Ac2O and AcOH, whereby the side-chain hydroxyl group is alkylthioalkylated. The reaction can be performed at an appropriate temperature (for example, 10 to 60° C.) for a predetermined time. The alkylthioalkylated product can be recovered as, for example, a precipitate. Furthermore, the resulting precipitate can also be dissolved in an appropriate solvent such as ethyl acetate, followed by sequential washing with an aqueous saturated sodium bicarbonate solution and an aqueous saturated sodium chloride solution, and the like. The resulting alkylthioalkylated product is subjected to appropriate treatments such as drying and concentration, followed by separation and purification. For the separation and purification, it is preferable to use, for example, a silica-gel chromatography.
- Next, when a converted disulfide is obtained from the alkylthioalkylated product, the following procedures are performed.
- The separated and purified alkylthioalkylated product is dissolved in a basic solvent containing a neutralizing agent, preferably N,N-diisopropylethylamine (DIEA) at an appropriate temperature (for example, −10 to 10° C.) for a predetermined time to neutralize it, then the resultant product is mixed with a solvent containing a halogenating agent, preferably sulfuryl chloride at an appropriate temperature (for example, 10 to 60° C.) for a predetermined time to halogenate the alkylthioalkyl group, and after that the resulting product is mixed with a solvent containing an agent that converts to a thiol group, preferably potassium thiotosylate at an appropriate temperature (for example, 10 to 60° C.) for a predetermined time to substitute thiol for the halogen. To the resulting produce is added But-SH, and the mixture is stirred at an appropriate temperature (for example, 10 to 60° C.) for a predetermined time, which is diluted with a proper organic solvent to obtain a converted disulfide. An organic layer containing the obtained converted disulfide may also be washed with a proper aqueous solvent (for example, an aqueous saturated sodium bicarbonate solution, an aqueous sodium chloride solution, and the like). The converted disulfide is subjected to proper procedures such as drying and concentration, followed by separation and purification. For the separation and purification, it is preferable to use, for example, a silica-gel chromatography. A converted trisulfide can be obtained from the alkylthioalkylated product in the same manner as above.
- The converted disulfide is dissolved in TFA, preferably TFA containing thioanisole at an appropriate temperature (for example, 10 to 60° C.) for a predetermined time. The thioanisole in the TFA can trap But cations, which may be derived from a decomposition of But ester. The obtained serine or threonine derivative is subjected to proper procedures such as drying and concentration, followed by separation and purification. For the separation and purification, it is preferable to use, for example, a silica-gel chromatography.
- Among of examples of the serine or threonine derivative, compounds of the above-mentioned
formula 1 wherein Y1 is a t-butyl group, Y2 is 9-fluorenylmethoxycarbonyl group, R1 is a hydrogen atom or a methyl group, R2 is a methylene group, and n is 2 are preferable. - The second peptide can be synthesized by, for example, bonding the serine or threonine derivative to the N-terminal end of the peptide chain which has been extended by using a peptide automatic synthesizer. For example, an amino acid residue bonded to a resin is used as a starting material, and the peptide chain length is extended using an automatic synthesizer according to an Fmoc method. Then, the serine or threonine derivative (for example, Fmoc-Ser(CH2—S—S-But)—OH) is added thereto, whereby it is condensed to the N-terminal end of the resin-bonded peptide. After that, the terminal Fmoc in the obtained resin is removed, dried it, a mixed liquid of TFA:phenol:distilled water:thioanisole is added thereto, and the mixture is stirred. The liquid is removed by passing nitrogen gas, to which diethyl ether is added to precipitate a peptide. After the precipitate is dried under vacuum, the peptide is extracted with an aqueous acetonitrile solution, and the resin is filtered. Purification with a reversed phase HPLC can provide a second peptide. The synthesis reaction of the second peptide may be performed at a proper temperature (for example, 10 to 60° C.) for a predetermined time. In the extension of the peptide chain, an amino acid residue modified with sugar or the like may be introduced in a conventional technique. For example, a resin whose peptide chain length is extended, and a sugar-modified amino acid protected with Fmoc are added to a solution of HBTU-HOBt/DMF and DIEA in N-methylpyrrolidone (NMP), and the reaction is performed at a proper temperature (for example, 50° C.) for a predetermined time, whereby the sugar-modified amino acid residue can be added on the peptide chain. Examples of the thus obtained second peptide may include
peptide 13 described above. - The synthesis of the first peptide is not particularly limited as long as a peptide containing, at the C-terminal end, an amino acid derivative having a thioester group can be obtained. The synthesis can be performed according to, for example, a direct synthesis method using a Boc method (Hojo, H.; Aimoto, S. Bull. Chem. Soc. Jpn., 64, 111-117, (1991)); a direct synthesis method using an Fmoc method (Li, X.; Kawakami, T.; Aimoto, S. Tetrahedron Lett., 39, 8669-8672, (1998)); a method in which a peptide chain is constituted using an Fmoc method, and then the C-terminal end is thioesterified (Shin, Y.; Winans, K. A.; Backes, B. J.; Kent, S. B. H.; Ellman, J. A.; Bertozzi, C. R. J. Am. Chem. Soc., 121, 11684-11689, (1999)); or the like. In addition, the synthesis can also be performed in a method described in International Patent Application No. PCT/JP2007/069555. A method described in WO2008/044628 will be explained below.
- According to the method described in WO2008/044628, a peptide containing, at the C-terminal side, an amino acid derivative having a thioester group can be synthesized through following steps (i) to (iv).
- In step (i), the deprotection of the Fmoc group is performed by, for example, treating an Fmoc-CLEAR amide resin with piperidine/N-methylpyrrolidone (NMP), and the resulting resin is caused to react with Fmoc-N(R)—CH(CH2SY)—C(═O)OH in HOBt/NMP and N,N′-dicyclohexyl carbodiimide (DCC)/NMP. The reaction may be performed at an appropriate temperature (for example, 40 to 60° C.).
- The group R in Fmoc-N(R)—CH(CH2SY)—C(═O)OH is an alkyl group having 1 to 12 carbon atoms or an aralkyl group having 7 to 12 carbon atoms. Examples of the alkyl group can include a methyl group, an ethyl group, a propyl group, a butyl group, a pentyl group, a hexyl group, and the like, which may be linear, branched or cyclic hydrocarbon groups. Examples of the aralkyl group can include a benzyl group, and the like. R is preferably a methyl group, an ethyl group, an isobutyl group or a benzyl group.
- Step (ii) is a step in which after the deprotection of the Fmoc group in the Fmoc-(amino acid residue)n-N(R)—CH(CH2SY)—C(═O)NH-resin in which n is 0 or 1, obtained in step (i), is performed, the resulting product is caused to react with an Fmoc-amino acid, if necessary, to produce an Fmoc-(amino acid residue)n-N(R)—CH(CH2SY)—C(═O)NH-resin in which n is 1 or 2, and then the deprotection of the Fmoc group and the reaction with the Fmoc-amino acid are repeated to produce an X—N(R)—CH(CH2SY)—C(═O)—NH-resin. When in step (i), the Fmoc-(amino acid residue)n-N(R)—CH(CH2SY)—C(═O)NH-resin is produced by causing the Fmoc-(amino acid residue)n-N(R)—CH(CH2SY)—C(═O)OH (n is 1) to react, the Fmoc-amino acid may be caused to react or not in step (ii).
- In step (ii), specifically, the reaction product from step (i) is washed with NMP, and then the resin is shaken in Ac2O-DIEA/NMP for 5 minutes. After washing with NMP and the removal of the Fmoc group with piperidine/NMP are performed, a solution of Fmoc-Gly and hexafluorophosphate, O-(7-azabenzotriazole-1-yl)-N,N,N′,N′-tetramethyluronium (HATU), and N,N-diisopropylethylamine (DIEA) dissolved in NMP is added thereto, and the reaction is performed. The reaction may be performed at an appropriate temperature (for example, 40 to 60° C.) for a predetermined time. Next, after the reaction product is washed with NMP, the dehydration of the Fmoc group and reaction with the Fmoc-amino acid are repeated by using a conventional peptide synthesizer in accordance with, for example, a Fast Moc method, whereby the peptide chain length can be extended.
- Step (iii) is a step in which the X—N(R)—CH(CH2SY)—C(═O)—NH-resin obtained in step (ii) is caused to react with a deprotecting agent (for example, trifluoroacetic acid, and the like), whereby the elimination from the resin and deprotection of the thiol group are performed. Specifically, Reagent K is added to the product (resin) in step (ii), and the reaction can be performed at room temperature. Here, Reagent K refers to a reagent of TFA:H2O:thioanisole:ethanedithiol:phenol=82.5:5:5:2.5:5.
- Step (iv) is a step in which the compound obtained in step (iii) is caused to react with an acidic thiol to produce a thioester compound. Examples of the acidic thiol to be used here may include mercaptocarboxylic acid and a mixture of mercaptan and a carboxylic acid. HSCH2CH2COOH(MPA), HSC6H4CH2COOH(MPAA), or a mixture of thiophenol and acetic acid can be more preferably used.
- In step (iv), TFA is removed from the product from step (iii) through nitrogen stream, to which an ether is added to generate a precipitate. The precipitate is washed with an ether, and then is dried. The crude peptide is extracted with an aqueous acetonitrile solution containing TFA, which is diluted with an aqueous solution of acetonitrile of acidic thiol such as an aqueous solution of 3-mercaptopropionic acid or 4-mercaptophenylacetic acid, and the resulting product is allowed to stand for several hours to several ten hours, whereby a peptide thioester compound represented by X—S—CH2CH2COOH or X—S—C6H4—CH2COOH can be obtained.
- Examples of the first peptide obtained through steps (i) to (iv) can include peptide 14 described above.
- In addition, a compound represented by Z—N(R)—CH(CH2SY)—C(═O)OH wherein Z is a hydrogen atom or a 9-fluorenylmethoxycarbonyl group, R is an alkyl group having 1 to 12 carbon atoms or an aralkyl group having 7 to 12 carbon atoms, and Y is a protecting group of thiol, R and Y being described in detail above, is synthesized as follows:
- First, a compound represented by YSCH2CH(NH2)C(═O)OH wherein Y is a protecting group of thiol group or cysteine in which the thiol group is protected, and a compound represented by R1CHO wherein R1 is a hydrogen atom, an alkyl group having 1 to 11 carbon atoms or an aryl group having 6 to 11 carbon atoms are caused to react to produce a compound represented by YSCH2CH(N═CHR1)C(═O)OH wherein Y and R1 are as defined above. Here, the alkyl group in R1 is preferably an alkyl group having 1 to 9 carbon atoms, more preferably an alkyl group having 1 to 6 carbon atoms. Examples thereof may include a methyl group, an ethyl group, a propyl group, a butyl group, a pentyl group, a hexyl group, and the like, which may be any of linear, branched or cyclic hydrocarbons. Particularly preferable R1s are a methyl group, an isopropyl group and a phenyl group. The reaction thereof with the compound represented by R1CHO can be performed by dissolving the cysteine in which the thiol group is protected in water containing ethanol and potassium hydroxide, adding the compound represented by R1CHO thereto, and stirring the mixture at an appropriate temperature (for example, room temperature) for a predetermined time.
- Subsequently, the compound represented by YSCH2CH(N═CHR1)C(═O)OH wherein Y and R1 are as defined above is caused to react with a hydrogenating agent (for example, NaBH4 or NaBH3CN) to produce a compound represented ,by YSCH2CH(NHCH2R1)C(═O)OH wherein Y and R1 are as defined above, and, if necessary, the resulting product is caused to react with 9-fluorenylmethoxycarbonyl-N-hydroxysuccinimide ester (hereinafter “Fmoc-OSu”) to protect the amino group with Fmoc group, whereby a compound represented by Z—N(R)—CH(CH2SY)—C(═O)OH wherein Z is a hydrogen atom or a 9-fluorenylmethoxycarbonyl group, R is a alkyl group having 1 to 12 carbon atoms, or an aralkyl group having 7 to 12 carbon atoms, and Y is a protecting group of thiol group can be produced. The hydrogenating agent may be caused to react in the presence of a base such as sodium hydroxide. Specifically, the hydrogenating agent (for example, NaBH4 or NaBH3CN) is added to the compound represented by YSCH2CH(N═CHR1)C(═O)OH, and the mixture is stirred at a proper temperature, whereby the reaction can be performed. When NaBH4 is used, it is preferable to use it as a solution thereof dissolved in an aqueous sodium hydroxide solution; whereas when NaBH3CN is used, a base is not particularly necessary. Furthermore, when the amino group is protected with the Fmoc group by causing it to react with Fmoc-OSu, the amino group can be protected with the Fmoc group by causing it to react with the Fmoc group in the presence of sodium carbonate in a proper solvent (for example, 1,2-dimethoxyethane).
- The ligation reaction of the first peptide and the second peptide can be performed in the presence of a reducing agent in a buffer solution or a mixed solution of a buffer and an organic solvent.
- The reducing agent is not particularly limited, as long as it can reduce the thiol auxiliary group in the serine or threonine derivative to a state having a free thiol group. It is preferable to use, for example, a water-soluble phosphine such as triscarboxyethyl phosphine (TCEP) or trishydroxymethyl phosphine, trimethyl phosphine, triethyl phosphine, triisopropyl phosphine, tributyl phosphine, triphenyl phosphine, or the like; it is more preferable to use water-soluble phosphine, further more preferable to use TCEP. When a thiol such as thiophenol, mercaptophenylacetic acid, t-butyl mercaptan or dithiothreitol is used as a reducing agent, the ligation efficiency may highly likely lowers, because of occurrence of thioester exchange. The concentration of the reducing agent can be suitably adjusted depending to the kind of the reducing agent, as long as it can reduce the thiol auxiliary group in the serine or threonine derivative to a state having the free thiol group. For example, when TCEP is used as the reducing agent, the concentration is preferably from 7.5 to 50 mM, more preferably from 15 to 30 mM.
- The buffer solution to be used in the ligation reaction of the first peptide and the second peptide can include phosphate buffer solution, Tris-hydrochloric acid buffer solution, citrate buffer solution, and the like. Among these, the phosphate buffer solution is preferable. The mixed solution of the buffer solution and the organic solvent used in the ligation reaction of the first peptide and the second peptide can include mixed solutions of the buffer solution described above and an organic solvent such as acetonitrile, DMF, or NMP. Among these, the mixed solution of phosphate buffer solution and acetonitrile is preferable. The concentrations of the buffer solution and the mixed solution of buffer solution and organic solvent, and the mixing ratio of the mixed solution can be suitably adjusted depending on the addition amounts of the first peptide used and the second peptide used, and the kinds of the buffer solution and the organic solvent. For example, when the addition amounts of the first peptide and the second peptide are both 20 nmol, the concentration of the phosphate buffer solution used in the ligation reaction is preferably from 0.1 to 1.0 M, more preferably from 0.1 to 0.5 M, and further more preferably from 0.1 to 0.2 M. Moreover, when the addition amounts of the first peptide and the second peptide are both 20 nmol, the mixing ratio between the 0.1 M phosphate buffer solution and acetonitrile in the mixed solution used in the ligation reaction is preferably 1:1 of 0.1 M phosphate buffer solution and acetonitrile.
- The ligation reaction is performed at a pH of preferably 5 to 9, more preferably 6 to 8, and further more preferably 7±0.2. The temperature and the time of the ligation reaction can be suitably adjusted. For example, the reaction temperature can be set at room temperature (10 to 40° C.) and the reaction time can be set to a time during which the ligated product of the first peptide and the second peptide can be obtained in a predetermined amount.
- In order to confirm the ligated product of the first peptide and the second peptide, a conventional method used for confirming a peptide can be used without any limitation, and for example, a method using an HPLC is preferable.
- The yield of the ligated product of the first peptide and the second peptide is measured as follows:
- The ligated product of the first peptide and the second peptide is subjected to an HPLC measurement by using a C18 column, and an aqueous acetonitrile solution containing 0.1% (v/v) TFA as an eluent. The yield is calculated by dividing a peak area of a desired product at 280 nm by the total of the peak area of desired product, a peak area of an unreacted C-terminal segment, and a peak area of a by-product derived from the C-terminal end.
- The present invention will be explained in more detail by means of Examples below, but the invention is not limited to the Examples.
- To Fmoc-Ser-OBut (2.0 g, 5.2 mmol) were added DMSO (10 ml, 140 mmol), Ac2O (6.6 ml, 70 mmol) and AcOH (10 ml, 180 mmol), and the mixture was caused to react at room temperature for two days. After vacuum concentration was carried out, distilled water was added thereto until precipitate was generated, and supernatant was removed therefrom. The precipitate was dissolved in ethyl acetate, which was washed with an aqueous saturated sodium bicarbonate solution and an aqueous saturated sodium chloride solution, and the resultant precipitate was dried with sodium sulfate. After the sodium sulfate was filtered off, vacuum concentration was carried out to give an oily material, and the oily material was purified through a silica gel column chromatography (silica gel 230 g, toluene:ethyl acetate=9:1) to obtain compound 15 (1.8 g, 4.1 mmol, 79%).
- Rf 0.48 (toluene:ethyl acetate=5:1). 1H-NMR (400 MHz, CDCl3, TMS), δ 7.76 (d, 2H, J=7.3 Hz, Ar), 7.61 (m, 2H, Ar), 7.40 (t, 2H, J=7.3 Hz, Ar), 7.33 (t, 2H, J=7.3 Hz, Ar), 5.63 (d, 1H, J=8.3 Hz, NH), 4.69 (d, 1H, J=11.7 Hz, O—CH2—S), 4.60 (d, 1H, J=11.2 Hz, O—CH2—S), 4.44 (m, 1H, αH), 4.39 (m, 2H, Fmoc CH2), 4.24 (brt, 1H, J=6.8 Hz, Fmoc CH), 3.99 (dd, 1H, J=3.4, 9.3 Hz, βH), 3.73 (dd, 1H, J=2.9, 9.3 Hz, βH), 2.12 (s, 3H, S—CH3), 1.46 (s, 9H, But). HRFABMS: calcd for C24H29NO5Sna 466.1664. Found: m/z 466.1632. [α]D=+0.56° (c=1.0, CHCl3).
-
Compound 15 described above (0.67 g, 1.5 mmol) and DIEA (0.31 ml, 1.8 mmol) were dissolved in dichloromethane (5.0 ml), and then the resulting solution was cooled to 0° C. Dichloromethane (0.5 ml) in which SO2Cl2 (0.15 ml, 1.9 mmol) was dissolved was added thereto, and then the resulting solution was stirred at room temperature for one hour. After that, DMF (0.5 ml) in which potassium thiotosylate (0.51 g, 2.3 mmol) was dissolved was added thereto, and the mixture was stirred at room temperature for 30 minutes. tBu-SH (0.34 ml, 4.7 mmol) was added thereto, and the mixture was stirred at room temperature. The resulting mixture was diluted with dichloromethane, and an organic layer was washed with an aqueous saturated sodium bicarbonate solution and an aqueous sodium chloride solution, which was then dried with anhydrous calcium chloride. After the calcium chloride was filtered off, the solvent was distilled away under a reduced pressure, and the residue was purified through a silica gel column chromatography (toluene-ethyl acetate, 15:1) to obtain compound 16 (0.53 g, 1.0 mmol, 68%). - Rf 0.51 (toluene:ethyl acetate, 9:1). 1H-NMR (400 MHz, CDCl3, TMS), δ 7.75 (d, 2H, J=7.3 Hz, Ar), 7.63-7.60 (m, 2H, Ar), 7.39 (brt, 2H, J=7.3 Hz, Ar), 7.30 (brt, 2H, J=7.6 Hz, Ar), 5.65 (d, 1H, J=8.3 Hz, NH), 4.85 (d, 1H, J=11.2 Hz, O—CH2—S), 4.75 (d, 1H, J=11.2 Hz, O—CH2—S), 4.44 (m, 1H, αH), 4.23 (brt, 1H, J=7.3 Hz, Fmoc CH), 4.05 (dd, 1H, J=2.9, 9.3 Hz, βH), 3.81 (dd, 1H, J=2.7, 9.3 Hz, βH), 1.49 (s, 9H, But), 1.31 (s, 9H, But). HRFABMS: calcd for C27H35NO5S2Na 540.1854. Found: m/z 540.1744. [α]D=+13.77° (c=1.3, CHCl3)
-
Compound 16 described above (110 mg, 0.21 mmol) was dissolved in TFA (1.0 ml) containing 5% thioanisole, and the mixture was allowed to stand at room temperature for 30 minutes. After the solvent was distilled away under a reduced pressure, the obtained residue was purified through a silica gel column chromatography (chloroform-methanol, 15 : 1) to obtain compound 10 (60 mg, 0.13 mmol, 61%). - Rf 0.46 (chloroform-methanol, 9:1 containing 1% acetic acid). 1H-NMR (400 MHz, CD3SOCD3), δ 7.88 (d, 2H, J=7.8 Hz, Ar), 7.72 (d, 2H, J=7.3 Hz, Ar), 7.41 (brt, 2H, J=7.5 Hz, Ar), 7.34 (brt, 2H, J=7.5 Hz, Ar), 4.86 (d, 1H, J=11.2 Hz, O—CH2—S), 4.81 (d, 1H, J=10.7 Hz, O—CH2—S), 3.83-3.74 (m, 2H, βH), 1.26 (s, 9H, But). HRFABMS: calcd for C23H27NO5S2Na 484.1228. Found: m/z 484.1244. [α]D=+26.9° (c=0.75, CHCl3).
- A Lys(Boc)-Lys(Boc)-Pro-Tyr(But)-Ile-Leu-CLEAR Acid resin was obtained from a starting material, Fmoc-Leu-CLEAR Acid resin (0.49 meq/g, 200 mg, 0.1 mmol) by the use of an automatic synthesizer according to a FastMoc method. To one third (0.033 mmol) of the amount of the resin obtained was added a solution of
compound 12 or Fmoc-Thr(GaINAc)—OH (48 mg, 0.066 mmol), 0.45 M HBTU-HOBt/DMF (138 μl, 0.063 mmol) and DIEA (23.0 μl, 0.132 mmol) dissolved in NMP, and the mixture was caused to react at 50° C. for one hour. Furthermore, the resulting product was condensed with Fmoc-Ala-OH (33 mg, 0.1 mmol) and then Fmoc-Asn(Trt)-OH (59.7 mg, 0.1 mmol) by using 1 M DCC (150 μl) and 1 M HOBt (150 μl). Finally, the resulting product was condensed with Fmoc-Ser(CH2—S—S-But)—OH (31 mg, 0.066 mmol) by using 1 M DCC (99 μl) and 1 M HOBt (99 μl). After a half amount (0.017 mmol) of the obtained resin, in which the terminal Fmoc was removed, was dried, liquid (1 ml) of TFA:phenol:distilled water:thioanisole=85:5:5:5 was added thereto, and the mixture was stirred for 30 minutes. The liquid was removed therefrom with nitrogen gas, to which diethyl ether was added to precipitate a peptide. This procedure was repeated three times, and the precipitates were dried under vacuum. The peptide was extracted with a 50% aqueous acetonitrile solution, and the resin was filtered off. Purification by using a reversed phase HPLC was made to obtain a desired peptide 13 (5.5 mg, 3.6 yield : 21%). - MALDI-TOF MS:found:m/z 1562.01 (M+H)+, calcd for m/z1561.80 (M+H)+. Amino acid analysis: Asp0.96Thr0.85Ser0.90Pro1.04Ala1.00Ile0.96Leu1.06Tyr0.98Lys1.55.
- (5) Synthesis of Peptide 14 (pGlu-Ser-Glu-Glu-Gly-Gly-SC6H4CH2COOH)
- After the Fmoc-CLEAR-Amide resin (0.46 meq/g, 440 mg, 0.2 mmol) was treated with 20% piperidine/NMP (5 minutes×one time, 15 minutes×one time), and washed with NMP (one minute×5 times), a solution obtained by stirring the Fmoc-(Et)Cys(Trt)-OH (250 mg, 0.4 mmol), 1 M DCC/NMP (600 μl) and 1 M HOBt/NMP (600 μl) at room temperature for 30 minutes was added thereto, and the mixture was shaken at 50° C. for 1 hour. After the Fmoc was eliminated with piperidine (5 minutes×one time, and 15 minutes×one time), a NAV solution in which Fmoc-Gly-OH (300 mg, 1.0 mmol), HATU (380 mg, 1.0 mmol) and DTFA (350 μl, 2.0 mmol) were dissolved was added thereto, and the mixture was shaken at 50° C. for 1 hour. This reaction was repeated once again. After a half amount (0.1 mmol) of the resin was taken out, the remaining resin was subjected to a treatment using an Applied Biosystems (
ABI 433A) automatic synthesizer to extend the peptide chain in accordance with a FastMoc method, whereby an Ser(But)-Glu(OBut)-Glu(OBut)-Gly-CLEAR amide resin was obtained. The resin was condensed with Boc-pGlu-OH (46 mg, 0.20 mmol) using 0.45 M HBTU, HOBt/DMF (420 0.19 mmol) and DIEA (70 0.40 mmol) to obtain Boc-pGlu-Ser(But)-Glu(OBut)-Glu(OBut)-Gly-CLEAR amide resin (330 mg). The whole amount of the resin was treated with Reagent K (TFA:phenol:distilled water:thioanisole : 3,6-dioxa-1,8-octanedithiol=82.5:5:5:5:2.5) (4.0 ml) for 2 hours. TFA was removed with nitrogen gas, to which diethyl ether was added to precipitate a peptide. This procedure was repeated three times, and the precipitate was dried under a reduced pressure. The precipitate was dissolved in a 30% aqueous acetonitrile solution (10 ml) containing 6 M urea, to which mercaptophenylacetic acid (0.2 ml) was added, and the mixture was caused to react overnight. After the resin was filtered off, a crude peptide was purified through a reversed phase HPLC to obtain peptide 14 (4.3 mg, 5.8 μmol, 6%). - MALDI-TOF MS:found:m/z 739.137 (M+H)+, calcd for: m/z 739.225 (M+H)+. Amino acid analysis : Ser0.75Glu2.63Gly2.01.
- (6) Synthesis of Peptide 17 (pGlu-Ser-Glu-Glu-Gly-Gly-Ser-Asn-Ala-Thr(GalNAcBn)-Lys-Lys-Pro-Tyr-Ile-Leu-OH) (see Table 1 described above)
- According to
condition 1 in Table 1,peptide 13 and peptide 14 (20 nmol, each) were dissolved in a 0.1 M sodium phosphate buffer (pH 7.2, 4 μl) containing 6 M guanidine hydrochloride and 0.2 M MPAA, and the mixture was allowed to stand at room temperature overnight. According tocondition 2 in Table 1,peptide 13 and peptide 14 (20 nmol, each) were dissolved in a 0.1 M sodium phosphate buffer (pH 7.0, 4 μl) containing 7.5 mM triscarboxyethyl phosphine (TCEP), and the mixture was allowed to stand at room temperature overnight. According tocondition 3 in Table 1,peptide 13 and peptide 14 (20 nmol, each) were dissolved in 50% 0.1 M sodium phosphate buffer-acetonitrile (pH 7.0, 4 μl) containing 7.5 mM triscarboxyethyl phosphine (TCEP), and the mixture was allowed to stand overnight. According tocondition 4 in Table 1,peptide 13 and peptide 14 (20 nmol, each) were dissolved in 50% 0.1 M sodium phosphate buffer-acetonitrile (pH 7.0, 4 μl) containing 7.5 mM triscarboxyethyl phosphine (TCEP), and DMF was added thereto after 10 minutes, and the mixture was allowed to stand overnight. - Fmoc-Thr-OBut (1.3 g, 3.3 mmol) was dissolved in DMSO (6.0 ml, 85 mmol), Ac2O (4.0 ml, 42 mmol) and AcOH (6.0 ml, 0.10 mol), and the mixture was caused to react at room temperature for two days. After vacuum concentration, distilled water was added thereto until precipitate was generated, and a supernatant was removed therefrom. The precipitate was dissolved in ethyl acetate, the resulting solution was transferred to a separating funnel, and after that, was washed with an aqueous saturated sodium bicarbonate solution and an aqueous saturated sodium chloride solution. After the ethyl acetate layer was dried with sodium sulfate, followed by filtration, and vacuum concentration was made to obtain an oily material. The oily material was purified through a silica gel column chromatography (silica gel 130 g, toluene:ethyl acetate=9:1) to obtain compound 18 (1.4 g, 3.0 mmol, 91%). Rf 0.49 (toluene:ethyl acetate=7:1). 1H-NMR (400 MHz, CDCl3, TMS), d 7.75 (d, 2H, J=7.8 Hz, Ar), 7.62 (m, 2H, Ar), 7.39 (brt, H, J=7.5 Hz, Ar), 7.31 (t, 2H, J=7.3 Hz, Ar), 5.50 (d, 1H, J=9.8 Hz, NH), 4.65 (d, 1H, J=11.7 Hz, O—CH2—S), 4.58 (d, 1H, J=11.7 Hz, O—CH2—S), 4.35 (m, 1H, bH), 4.27 (dd, 1H, J=2.0, 9.8 Hz, aH), 4.24 (brt, 1H, J=6.8 Hz, Fmoc CH), 2.13 (s, 3H, SCH3), 1.49 (s, 9H, But), 1.23 (d, 3H, J=6.3 Hz, Thr CH3). [a]D=−19.1° (c 1.0 in Chloroform). MALDI-TOF MS: calcd for C25H31NNaO5S 480.182. Found: m/z 480.054.
- After compound 2 (0.50 g, 1.1 mmol) was azeotroped with toluene to dehydrate it and DCM (4.5 ml) was added thereto, DIEA (0.20 ml, 1.1 mmol) was added to the mixture to make it basic. After the mixture was cooled with ice, sulfuryl chloride (92 μl, 1.1 mmol) was added thereto, and the reaction was performed for 30 minutes, and then the temperature was returned to an ordinary temperature. Potassium thiotosylate (0.38 g, 1.7 mmol) dissolved in DMF (2.3 ml) was added to the mixture, and the reaction was performed for 30 minutes, to which t-butyl mercaptan (0.27 ml, 2.4 mmol) was added, and the reaction was performed for 30 minutes. Ethyl acetate was added to the reaction mixture, which was washed with a 1 M aqueous saturated HCl solution and then was dried with sodium sulfate. The resulting product was filtered and then, after vacuum concentration, purified through a silica gel column chromatography (90g, hexane:ethyl acetate=4:1) to obtain compound 19 (0.48 g, 0.90 mmol, 82%).
- Rf 0.38 (hexane:ethyl acetate=4:1). 1H-NMR (400 MHz, CDCl3, TMS), d 7.75 (d, 2H, J=7.3 Hz, Ar), 7.62 (m, 2H, Ar), 7.39 (brt, H, J=7.6 Hz, Ar), 7.31 (brt, 2H, J=7.6 Hz, Ar), 5.49 (d, 1H, J=9.8 Hz, NH), 4.88 (d, 1H, J=11.2 Hz, O—CH2—S), 4.75 (d, 1H, J=11.2 Hz, O—CH2—S), 4.44 (m, 1H, bH), 4.28 (dd, 1H, J=2.4, 9.7 Hz, aH), 4.24 (brt, 1H, J=7.3 Hz, Fmoc CH), 1.51 (s, 9H, But), 1.33 (s, 9H, But), 1.27 (d, 3H, J=6.3 Hz, Thr CH3). [a]D=19.1° (c 1.0 in Chloroform). MALDI-TOF MS: calcd for C28H37NNaO5S2 554.201. Found: m/z 554.386.
- Compound 5 (0.48 g, 0.90 mmol) was dissolved in 50% TFA-dichloromethane (5.0 ml) containing 5% thioanisole, and the reaction was performed for 30 minutes. After the reaction was finished, the reaction product was purified through a silica gel column chromatography (60 g, chloroform:methanol:acetic acid=15:1:0.1), and then through a silica gel column chromatography (40g, toluene:ethyl acetate:acetic acid=2:1:0.1). After that, the product was purified through a gel filtration chromatography to obtain compound 11 (95 mg, 0.20 mmol, 22%).
- Rf 0.39 (chloroform:methanol: acetic acid=5:1:0.1). 1H-NMR (400 MHz, CDCl3, TMS), d 8.50 (brs, 1H, OH), 7.74 (d, 2H, J=8.0 Hz, Ar), 7.60 (m, 2H, Ar), 5.59 (m, 1H, NH), 5.10-4.71 (m, 2H, O—CH2—S), 4.50-4.36 (m, 4H, aH, bH, Fmoc CH2), 4.23 (t, 1H, J=6.8 Hz, Fmoc CH), 1.41-1.14 (m, 12H, But, Thr CH3). [a]D=−41.5° (c 1.0 in Chloroform). MALDI-TOF MS: calcd for C24H29NNaO5S2 498.139. Found: m/z 498.084.
- According to the method for producing a peptide of the present invention, a protein or glycoprotein having high stability can be solution-phase synthesized in a high yield, while the generation of by-products is restrained. The protein or glycoprotein obtained by the method for producing a peptide of the invention can be utilized in a production of pharmaceutical products. Furthermore, the glycoprotein produced by the present invention can be expected to show the same activity as that of a naturally occurring glycoprotein, such as an activity of bonding to a specific cell receptor as one kind of nerve transmitter substances.
Claims (11)
1.-6. (canceled)
7. A method for producing a peptide which comprises a step of causing a first peptide and a second peptide to react in the presence of a reducing agent to obtain a ligated product of the first peptide and the second peptide, wherein the first peptide contains, at the C-terminal end, an amino acid derivative having a group represented by a formula: —CO—S—R
wherein R is an alkyl group having 1 to 12 carbon atoms which may be substituted by a carboxyl group, or an aryl group having 7 to 12 carbon atoms which may be substituted by a carboxyl group, as a thioester group; and
the second peptide contains, at the N-terminal end, a serine or threonine derivative having a group represented by a following formula 1:
8. The method for producing a peptide according to claim 7 , wherein the thiol auxiliary group is a group containing a disulfide bond wherein n is 2 in the formula (1).
9. The method for producing a peptide according to claim 7 , wherein the reducing agent is a phosphine compound.
10. The method for producing a peptide according to claim 7 , wherein Y1 is t-butyl group, Y2 is 9-fluorenylmethoxycarbonyl group, R1 is a hydrogen atom or a methyl group, R2 is a methylene group, and n is 2.
12. The compound according to claim 11 , wherein Y1 is a t-butyl group, Y2 is a 9-fluorenylmethoxycarbonyl group, R1 is a hydrogen atom or a methyl group, R2 is a methylene group, and n is 2.
13. The method for producing a peptide according to claim 8 , wherein the reducing agent is a phosphine compound.
14. The method for producing a peptide according to claim 8 , wherein Y1 is t-butyl group, Y2 is 9-fluorenylmethoxycarbonyl group, R1 is a hydrogen atom or a methyl group, R2 is a methylene group, and n is 2.
15. The method for producing a peptide according to claim 9 , wherein Y1 is t-butyl group, Y2 is 9-fluorenylmethoxycarbonyl group, R1 is a hydrogen atom or a methyl group, R2 is a methylene group, and n is 2.
16. The method for producing a peptide according to claim 13 , wherein Y1 is t-butyl group, Y2 is 9-fluorenylmethoxycarbonyl group, R1 is a hydrogen atom or a methyl group, R2 is a methylene group, and n is 2.
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JP2008-260938 | 2008-10-07 | ||
PCT/JP2009/005180 WO2010041425A1 (en) | 2008-10-07 | 2009-10-06 | Method for producing a peptide |
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EP (1) | EP2343309A4 (en) |
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2009
- 2009-10-06 JP JP2010532809A patent/JPWO2010041425A1/en not_active Ceased
- 2009-10-06 EP EP09818965A patent/EP2343309A4/en not_active Withdrawn
- 2009-10-06 US US13/122,850 patent/US20120004457A1/en not_active Abandoned
- 2009-10-06 WO PCT/JP2009/005180 patent/WO2010041425A1/en active Application Filing
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